Anesthetic and Cardio-Pulmonary Effects of Propofol Or Alfaxalone with Or Without Midazolam Co-Induction in Fentanyl Sedated Dogs by Penting Liao

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Anesthetic and Cardio-pulmonary Effects of Propofol or Alfaxalone with or without Midazolam Co-Induction in Fentanyl Sedated Dogs by PenTing Liao

A Thesis presented to The University of Guelph

In partial fulfilment of requirements for the degree of Doctor of Veterinary Science in Clinical Studies

Guelph, Ontario, Canada © PenTing Liao, August, 2016

ABSTRACT

Anesthetic and Cardio-pulmonary Effects of Propofol or Alfaxalone with or without Midazolam Co-Induction in Fentanyl Sedated Dogs For Diagnostic Imaging

PenTing Liao Advisor: University of Guelph, 2016 Dr. Melissa Sinclair

This thesis describes a prospective, randomized, incomplete Latin-square crossover, blind trial to investigate the effects of midazolam (M) as a co-induction agent in dogs induced and maintained with propofol (P) or alfaxalone (A) for diagnostic imaging. The quality of induction and recovery, induction and maintenance dose requirements for P or A, ease of maintenance using total intravenous anesthesia (TIVA), and cardio-pulmonary effects were determined in ten dogs assigned to P-S: P with saline (S); A-S: A with S; P-M: P with M;

A-M: A with M. Fentanyl (7 µg kg-1, IV) was administered 10 minutes prior to an IV bolus of

P (1 mg kg-1) or A (0.5 mg kg-1) followed by M (0.3 mg kg-1, IV) or S and additional boluses of P or A for intubation, followed by P or A TIVA during imaging.

The induction quality was significantly better in A-M versus A-S, P-M versus P-S, and A-M versus P-S. The induction dose was significantly lower in P-M versus P-S, and A-M versus A-S. The TIVA rate with P-M was significantly lower than P-S but similar between

A-M and A-S. Sedation, extubation and recovery quality, and TIVA duration were similar between treatments. Time to standing was significantly longer for A than P, but was similar within A or P treatments.

After induction, heart rate (HR) was significantly higher in A-M than A-S and P-S.

During imaging, HR of A-S and A-M were significantly higher than P-S. Before recovery,

HR of A-M was significantly higher than P-S. Systolic blood pressure of A-S was

significantly higher than A-M and P-M. There was no significant treatment difference for mean or diastolic blood pressure, cardiac index (CI), respiratory rate, occurrence of apnea, end-tidal CO2, and blood gas values. However, CI and HR significantly decreased after imaging compared to other phases.

Midazolam improved the quality and reduced the required dose for both P and A induction, and reduced TIVA rate of P. There was no significant cardiopulmonary difference identified between treatments despite co-induction with M. The decrease in CI and HR after imaging warrants close monitoring during recovery.

ACKNOWLEDGEMENTS

First, I would like to thank my dad, mom, brother and two sisters who support me unconditionally for the past 30 years.

Second, I would like to thank Drs. Melissa Sinclair, Alex Valverde, Conny Mosley and

Craig Mosley for their clinical and academic mentoring. Special thank to my supervisor Dr.

Sinclair who made the whole residency/DVSc training possible for me and Dr. Sawyer who encouraged and assisted me to apply in the first place.

Third, I would like to thank my resident mates Andrea, Alicia and Rodrigo for helping each other out whenever needed and learned from each other.

Fourth, I would like to thank all the veterinarians and technicians in OVC especially anesthesia section: Emily, Lucy, Cindy, Andrea, Ines, Rob, Megan, Nick, Jen, Shirley for their patience and kindness.

Fifth, I would like to thank my neighbors and resident mates Miranda, Gonzalo and

Rames whose presence made last three years more fun and pleasant.

Last, I would like to thank everyone I meet along the way of my life. I am who I am because of all of you.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS………………………………………………………………...iv

TABLE OF CONTENTS…………………………………………………………………....v

DECLARATION OF WORK PERFORMED…...………………………………...….…. x

LIST OF TABLES……………………………………………………...…………………. xi

LIST OF FIGURES……………………………………………………………………….xiii

CHAPTER I

GENERAL LITERATURE REVIEW……………………………………………………..…1

Introduction and objectives…………….……….………………………………………..…..1

Study goals and statement of hypothesis……..………………………………………….…..3

Literature review……………………………..………………………………………………4

Pre-anesthetic sedation…………………….………………………………………………...4

Injectable induction agents………………….……………………………………………….7

Alfaxalone……………………….……………………………………………….….7

Physical and chemical properties………….………………………….……..8

Pharmacology: Central nervous system effects………………………….….9

Cardiovascular effects…………………………………………………..…10

Respiratory effects………………………………………………………...12

Induction dosage and quality……………………………………………...14

Propofol…………………………………………………………………………...14

Physical and chemical properties………………………………………....14

Pharmacology: Central nervous system effects……………………….…..15

Cardiovascular effects………………………………………………….…15

Respiratory effects………………………………………………………...16

Induction dosage and quality………………………………………………17

The use of co-induction agents…………………………………………………….………18

Advantages…………………………………………………………………...…....18

Disadvantages…………………………………………………….………………..19

Drugs commonly used as co-induction agents…………………………………….19

Benzodiazepines…………………………………………………………...19

Lidocaine…………………………………………………………………..23

Ketamine…………………………………………………………………..24

Total intravenous anesthesia (TIVA)..……………………………………………………..25

History of TIVA……………………………………………………………...…....25

TIVA compared to inhalant anesthesia………………………………………….....26

Use of alfaxalone as a total intravenous agent…………………………………….27

Pharmacokinetics of alfaxalone………………………………………...…27

Cardiovascular and respiratory effects of alfaxalone TIVA…………….…27

Use of propofol as a total intravenous agent…………………………………...….28

Pharmacokinetics of propofol…………………………………………..…28

Cardiovascular and respiratory effects of propofol TIVA…………………28

Recovery characteristics from alfaxalone and propofol…………………………..29

Recovery quality of alfaxalone………………………………………...….29

Recovery time with alfaxalone………………………………………..…..29

Recovery quality of propofol…………………………………………..….30

Recovery time with propofol………………………………………….…..30

Comparison between alfaxalone and propofol for induction and maintenance…..31

Cardiac output (CO) measurement in research……………………………………………31

Definition and importance of cardiac output……………………………………...32

Techniques of cardiac output measurement………………………………………32

Direct Fick method………………………………………………….……33

Indicator method………………………………………………………….34

Pulse contour method……………………………………………….…….37

Ultrasound-based method…………………………………………………39

Thoracic electrical impedance method………………………………...…40

Validation in dogs…………………………………………………………………40

Direct Fick method…………………………………………………...…..40

Thermodilution method………………………………………………...…41

Lithium dilution method………………………………………………..…41

Pulse contour methods……………………………………………….……42

PulseCO…………………………………………………...………42

PiCCO…………………………………………………………..…43

FloTrac/Vigileo……………………………………………………43

Indirect Fick method………………………………………………………44

NICO………………………………………………………………44

Thoracic electrical impedance method……………………………………44

Ultrasound-based method…………………………………………………45

Summary of cardiac output methods…………………………………………...…46

Literature review summary……………………………………………………………..…47

References…………………………………………………………………………………48

CHAPTER II

INDUCTION DOSE AND RECOVERY QUALITY OF PROPOFOL AND ALFAXALONE

vii

WITH OR WITHOUT MIDAZOLAM CO-INDUCTION FOLLOWED BY TOTAL

INTRAVENOUS ANESTHESIA IN DOGS…………….………………………………..74

Summary………………………………………………………………………………..…74

Introduction……………………………………………………………………….………76

Materials and methods…………………………………………………………...….……78

Results……………………………………………………………………………...…..…85

Discussion…………………………………………………………………………………88

References……………………………………………………………………...…………97

Appendices……………………………………………………………………………….106

CHAPTER III

CARDIO-PULMONARY EFFECTS OF PROPOFOL OR ALFAXALONE WITH OR

WITHOUT MIDAZOLAM CO-INDUCTION FOLLOWED BY TOTAL INTRAVENOUS

ANESTHESIA IN FENTANYL SEDATED DOGS…………………………………….111

Summary………………………………………………………………………………...111

Introduction……………………………………………………………………………...113

Materials and methods………………………………………………………………..…115

Results…………………………………………………………………………………...123

Discussion……………………………………………………………………………….127

References…………………………………………………………………………….…131

CHAPTER IV

GENERAL DISCUSSION AND CONCLUSIONS……………………………………..145

Limitations of the study……………………………………………………………….…147

Strengths of the study…………………………………………………………………….149

viii

Areas for future research…………………………………………………………………150

References……………………………………………………………………………..…152

APPENDICES…………………………………………………………………………....154

DECLARATION OF WORK PERFORMED

The authors’ contribution is as follow:

PenTing Liao: Data collection, statistical analysis and manuscript preparation;

Melissa Sinclair: Study design and funding application, data collection, video scoring of recovery and manuscript review and revision; Alexander Valverde: Data collection, video scoring of recovery and manuscript review and revision; Conny Mosley: Manuscript review, video scoring of recovery and revision; Heather Chalmers: Manuscript review and revision;

Shawn Mackenzie: Data collection and manuscript review and revision; Brad Hanna:

Manuscript review and revision.

LIST OF TABLES

TABLE 1. SUMMARY OF CARDIOVASCULAR EFFECTS OF INDUCTION WITH ALFAXALONE IN

DOGS ...... 70

TABLE 2. INDUCTION DOSES AND QUALITY WITH ALFAXALONE ...... 72

TABLE 3. DESCRIPTIVE QUALITY SCORES FOR SEDATION (SEDQ) (0-NONE TO 3-PROFOUND),

TIME FROM PREMEDICATION TO INDUCTION (T2), INDUCTION QUALITY (INDQ) (0-SMOOTH

TO 4-NOT POSSIBLE), NUMBER OF ADDITIONAL DOSES DURING ANESTHETIC INDUCTION

(ADD-DOSE), AND TOTAL ANESTHETIC DOSE (TOTALD)...... 1033

TABLE 4. TOTAL INTRAVENOUS ANESTHESIA DOSES (TIVA), TIME FROM ADMINISTRATION OF

SEDATION TO TIVA DISCONTINUATION (T1), TOTAL ANESTHESIA TIME FROM THE START

OF INDUCTION TO TIVA DISCONTINUATION (T3), TIME FROM TIVA DISCONTINUATION TO

EXTUBATION (T4), TIME FROM TIVA DISCONTINUATION TO STANDING (T5), AND SIMPLE

DESCRIPTIVE ANESTHESIA MAINTENANCE QUALITY SCORES (ANESQ) (0-NO ADJUSTMENTS

TO 4-INTERVENTION REQUIRED) FOLLOWING INDUCTION WITH EITHER PROPOFOL (P) +

MIDAZOLAM (M) (PM); ALFAXALONE (A) + M: (AM); P + SALINE (S) (PS); AND A + S

(AS)...... 1044

TABLE 5. DESCRIPTIVE QUALITY SCORES OF EXTUBATION (EXTQ) (0-VERY CALM TO 4-

EXTREME EXCITEMENT) AND RECOVERY (RECQ) (0-EXCITABLE TO 3-PROFOUND SEDATION)

AFTER EXTUBATION FOLLOWING PROPOFOL (P) OR ALFAXALONE (A) TOTAL INTRAVENOUS

ANESTHESIA FOR DIAGNOSTIC IMAGING.… ...... 105

TABLE 6. MEDIAN AND 95% CONFIDENCE INTERVAL FOR SYSTOLIC (SAP) AND DIASTOLIC

(DAP) BLOOD PRESSURE...... 1388

TABLE 7. MEDIAN AND 95% CONFIDENCE INTERVAL FOR CARDIAC INDEX (CIL), STROKE

VOLUME INDEX (SVIL) AND SYSTEMIC VASCULAR RESISTANCE INDEX (SVRIL)

CALCULATED FROM CARDIAC OUTPUT (CO) MEASURED BY LIDCO...... 1400

TABLE 8. MEDIAN AND 95% CONFIDENCE INTERVAL FOR CARDIAC INDEX (CIP) CALCULATED

FROM CARDIAC OUTPUT (CO) MEASURED BY PULSECO...... 1411

TABLE 9. MEAN AND 95% CONFIDENCE INTERVALS (CI) FOR RESPIRATORY RATE (FR) AND

MEDIAN AND 95% CI END-TIDAL CARBON DIOXIDE (PE’CO2)...... 1422

TABLE 10. MEDIAN AND 95% CONFIDENCE INTERVAL FOR TEMPERATURE AND ARTERIAL

BLOOD GAS ANALYSIS, INCLUDING PH, ARTERIAL CARBON DIOXIDE (PACO2), OXYGEN

+ + - (PAO2) PARTIAL PRESSURE, SODIUM (NA ), POTASSIUM (K ), CHLORIDE (CL ),

HEMOGLOBIN (HB), AND LACTATE (LAC) CONCENTRATION...... 1433

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LIST OF FIGURES

FIGURE 1. STUDY TIMELINE WITH ADMINISTRATION OF FENTANYL (F); PROPOFOL (P) OR

ALFAXALONE (A) WITH EITHER SALINE (S) OR MIDAZOLAM (M) ...... 1022

FIGURE 2. STUDY TIMELINE WITH ADMINISTRATION OF FENTANYL (F); PROPOFOL (P) OR

ALFAXALONE (A) WITH EITHER SALINE (S) OR MIDAZOLAM (M) ...... 1355

FIGURE 3. MEDIAN AND 95% CONFIDENCE INTERVAL FOR (A) HEART RATE (HR), (B) MEAN

(MAP) ARTERIAL PRESSURE IN DOGS RANDOMLY ASSIGNED TO ONE OF THE FOUR

TREATMENTS: PROPOFOL (P) + MIDAZOLAM (M) (PM); ALFAXALONE (A) + M: (AM); P +

SALINE (S) (PS); AND A + S (AS)...... 1366

FIGURE 4. STUDY CONSORT DIAGRAM DEMONSTRATES SAMPLE NUMBERS IN EACH IMAGING

MODALITIES INCLUDING MAGNETIC RESONANCE IMAGING (MRI); COMPUTER

TOMOGRAPHY WITH INTERVERTEBRAL DISC INJECTION (CT1); COMPUTER TOMOGRAPHY

WITHOUT INTERVERTEBRAL DISC INJECTION (CT2) AND THE TREATMENTS DISTRIBUTION:

PROPOFOL (P) - SALINE (S); P – MIDAZOLAM (M); ALFAXALONE (A) – S; AM...... 152

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CHAPTER 1: GENERAL LITERATURE REVIEW 1

INTRODUCTION AND OBJECTIVES 2

General anesthesia is defined as a reversible, controlled, and drug-induced 3 unconsciousness which is not arousable by noxious stimulation (Tranquilli et al. 2007). There are 4 three main phases of general anesthesia: the induction phase, from consciousness to 5 unconsciousness to facilitate endotracheal intubation; the maintenance phase, requiring a 6 consistent surgical or diagnostic procedural depth with appropriate muscle relaxation and 7 analgesia without movement; and the recovery phase, with return to consciousness and 8 awareness. The induction of general anesthesia is commonly accomplished with a primary 9 injectable induction agent (such as propofol or alfaxalone) alone or in combination with 10 co-induction agents such as a benzodiazepine, lidocaine, or an opioid bolus. 11

Supplementary or co-induction agents are used with the primary injectable anesthetic 12 agent to promote a smooth induction to unconsciousness and endotracheal intubation and to 13 minimize the dose of the primary anesthetic induction agent, potentially reducing negative 14 cardiovascular effects. The main co-induction agents reported in veterinary medicine are fentanyl, 15 diazepam, midazolam, lidocaine, and low-dose ketamine. Data is available from both human and 16 veterinary research describing the possible injectable dose reduction with co-induction agents, 17 and includes both positive and negative findings (Short et al. 1992; Anderson & Robb 1998; 18

Braun et al. 2007; Robinson & Borer-Weir 2013; Sanchez et al. 2013). In veterinary medicine, 19 the ability to demonstrate a dose reduction of the primary induction agent is influenced by the 20 order of administration of the co-induction agent relative to the primary injectable induction 21 agent, especially with benzodiazepines (Sanchez et al. 2013) as well as the assessment methods 22 of the study. Other reported factors impacting the benefits of these supplementary agents in dogs 23

1 include the dose, interval and speed of injection of the induction and co-induction agent, and the 24 sedation level of the dog prior to induction (Covey-Crump & Murison 2008; Robinson & 25

Borer-Weir 2013; Sanchez et al. 2013). The research to date in dogs suggests that a dose of 26 midazolam of 0.2-0.4 mg kg-1, intravenous (IV), is ideal after an initial IV bolus of propofol 27

(Robinson & Borer-Weir 2013). The potential dose reduction, induction quality, and ease of 28 endotracheal intubation in dogs given midazolam and alfaxalone as co-induction agents have not 29 been investigated. 30

For propofol, the overall cardiovascular effects and benefit of combining the injectable 31 anesthetic with the co-induction agent remains controversial in dogs (Anderson & Robb 1998; 32

Jones et al. 2002; Goel et al. 2008; Hopkins et al. 2013). To the authors’ knowledge, scientific 33 studies investigating advanced cardiovascular and respiratory measurements during the induction 34 phase and endotracheal intubation in fentanyl pre-medicated dogs induced with either propofol 35 or alfaxalone, with or without midazolam co-induction, are not available. In addition, the ease of 36 maintenance, cardio-pulmonary effects, and recovery characteristics during diagnostic imaging 37 between propofol and alfaxalone have not been fully compared, especially with midazolam 38 co-induction. 39

The purpose of this research is to investigate the cardio-pulmonary effects, induction 40 dose reduction, quality during the induction phase, total intravenous anesthesia (TIVA) 41 maintenance dose, and recovery characteristics with propofol or alfaxalone, with or without 42 midazolam co-induction, in fentanyl sedated dogs. During the induction phase, total propofol or 43 alfaxalone dose requirements, number of additional injectable doses, induction quality scoring, 44 heart rate (HR), respiratory rate (RR), arterial blood pressure (ABP), cardiac index (CI), stroke 45 volume index (SVI), systemic vascular resistance index (SVRI), oxygen saturation percentage 46

(SPO2), and end-tidal carbon dioxide (ETCO2) will be compared between treatments prior to 47 mechanical ventilation. Maintenance quality, cardiovascular stability during propofol or 48 alfaxalone TIVA during mechanical ventilation, and recovery characteristics during MRI or CT 49 diagnostic imaging with or without intra-intervertebral disc injection will also be compared for 50 propofol and alfaxalone. 51

STUDY GOALS AND STATEMENT OF HYPOTHESIS 52

This study will focus on three main goals: 1) to investigate the dose reduction of propofol 53 and of alfaxalone for endotracheal intubation with and without midazolam co-administration in 54 fentanyl sedated dogs; 2) to investigate the cardio-pulmonary effects of induction with propofol 55 alone and with alfaxalone alone and when each is combined with midazolam in fentanyl sedated 56 dogs; and 3) to investigate and compare the cardiovascular effects, maintenance quality, and 57 recovery characteristics between alfaxalone and propofol TIVA with or without midazolam 58 co-induction during MRI or CT diagnostic imaging. 59

The hypotheses are as follows: 60

With or without co-induction with midazolam (0.3 mg kg-1, IV) after an initial bolus of propofol 61 or alfaxalone in fentanyl sedated dogs: 62

1) There is no difference in the total dose of the induction agent required for 63

endotracheal intubation 64

2) There is no difference in the induction quality and the requirements for additional 65

injectable boluses to allow for endotracheal intubation 66

3) There is no difference in the cardio-pulmonary effects and maintenance quality 67

during induction and TIVA during MRI or CT examination. 68

4) There is no difference in the extubation and recovery characteristics. 69

LITERATURE REVIEW 70

PRE-ANESTHETIC SEDATION 71

Pre-anesthetic sedation is important prior to induction of anesthesia to reduce the 72 anxiousness and stress of the patient, provide pre-emptive analgesia for surgery, help minimize 73 the amount of the injectable anesthetic agent required to induce unconsciousness, and also reduce 74 the injectable or inhalant requirements during the maintenance phase of anesthesia (Grimm et al. 75

2015). 76

Opioids are commonly used sedatives in cardiovascularly compromised patients because 77 of their high margin of safety, having minimal cardiopulmonary depression while providing 78 excellent analgesia (Tranquilli et al. 2007). The first documented medical use of opium traces 79 back to 2100 BC on the Sumerian clay tablet (Norn et al. 2005). Nevertheless, it wasn't until 80

1806 that morphine was isolated, followed by the development of other pure alkaloids and 81 eventual wide spread use in the middle of nineteenth century (Norn et al. 2005). 82

There are three well-recognized opioid receptors µ (mu), κ (kappa), and δ (delta). 83

According to the ligand receptor interaction, opioids can be classified into full agonists, partial 84 agonists, and antagonists to any one of the opioid receptors, or as agonist/antagonists that interact 85 with more than one receptor. Fentanyl is a pure mu-agonist with rapid onset, short duration, and 86 a wide margin of safety. Despite individual variability, it is typically given at 5-10 µg kg-1, IV, as 87 a bolus in canines for sedation and analgesia (Kamata et al. 2012; Kukanich & Clark 2012). 88

The pharmacokinetic profile of fentanyl following IV administration has been reported 89 in multiple scientific studies. Interestingly, one study demonstrated that fentanyl possesses a 90 dose-independent pharmacokinetic profile after single intravenous doses from 2.5-640 µg kg-1 91

(Murphy et al. 1983). In general, other anesthetic agents have minimal influence on fentanyl’s 92

4 pharmacokinetic profile. The impact of acepromazine (0.05 mg kg-1, IV) compared to 93 dexmedetomidine (2.5 µg kg-1, IV), administered in the recovery period have both been assessed 94 after 120 minutes of isoflurane general anesthesia with a constant rate infusion (CRI) of fentanyl 95

(5 µg kg-1 hr-1). This showed that acepromazine, but not dexmedetomidine, slightly increased 96 fentanyl’s clearance (128%) with minimal influence on plasma concentration compared to saline 97 treatment, when the CRI of fentanyl was continued for 60 minutes after isoflurane 98 discontinuation (Keating et al. 2015). In this study, systemic clearance of fentanyl ranged from 99

27.3-37.7 mL kg-1 min-1 and central and peripheral volume of distribution ranged from 0.69-0.81 100

L kg-1 to 3.17-4.17 L kg-1 (Keating et al. 2015). The reported elimination half-life, clearance, and 101 volume of distribution of fentanyl in anesthetized dogs ranges from 2.4-3.4 hours, 32.6-58.9 mL 102 kg-1 min-1 and 7.7-10.2 L kg-1 (Murphy et al. 1979; Lin et al. 1981) and in conscious dogs ranges 103 from 0.75-6 hours, 20-77.9 mL kg-1 min-1 and 4.7-10.7 L kg-1 (Kyles et al. 1996; Sano et al. 2006; 104

Little et al. 2008; KuKanich & Hubin 2010). However, the short duration of action seen with 105 fentanyl clinically is the result of rapid redistribution and lowering of plasma concentration. 106

Hence, elimination half-life is not a good indicator for actual drug duration if a low loading dose 107 has been administered. Due to its high lipophilicity, fentanyl crosses the blood brain barrier with 108 ease, with concentrations within the cerebrospinal fluid and brain tissue peaking within 2-10 109 minutes (Hug & Murphy 1979) and 10-15 minutes (Ainslie et al. 1979), respectively, after IV 110 administration. 111

Fentanyl has an incredibly high therapeutic index. No mortalities resulted when 13 dogs 112 were experimentally injected with 38.2 mg kg-1, IV (Bailey et al. 1987). However, these dogs 113 showed signs of sedation, such as ataxia and recumbency, and progressed to being non 114 responsive to tail-clamping for a variable duration. This safety is likely related to the fact that 115

5 fentanyl has minimal negative effects on the cardiovascular system (Kukanich & Clark 2012). At 116 a dose of 15 µg kg-1, IV, fentanyl significantly reduces heart rate, however, cardiac index (CI; 117 normalized cardiac output by body weight or surface area) and blood pressure are not 118 significantly altered in dogs (Salmenpera et al. 1994). Cardiac output and blood pressure 119 decreased moderately when fentanyl 50 µg kg-1, IV, was administered during enflurane 120 anesthesia (Hirsch et al. 1993). In another study, unsedated research dogs receiving a cumulative 121 dose of fentanyl of 27.5 µg kg-1, IV, over 15 minutes showed a mild increase in blood pressure 122

(Arndt et al. 1984). In contrast to what has been demonstrated in people, fentanyl has a 123 dose-dependent but minimal respiratory depressive effect at clinical doses in conscious dogs 124

(Grimm et al. 2005). Moreover, the respiratory depression evident shows a ceiling effect, with 125 only mild to moderate increases in arterial carbon dioxide observed at up to a hundred times the 126 clinical dose in dogs (Bailey et al. 1987). These favorable characteristics of fentanyl make it a 127 suitable and popular pre-medicant in cardiovascularly compromised patients. 128

In one study, fentanyl has been used as a co-induction agent in dogs. The dogs were 129 given a bolus of fentanyl at 2 µg kg-1, IV, prior to propofol induction to investigate the dose 130 reduction of propofol (Covey-Crump & Murison 2008). This resulted in a dose reduction in 131 propofol and overall improved induction characteristics when compared to midazolam 132 administration prior to propofol. The use of opioids as co-induction agents is more common in 133 people and has been demonstrated with fentanyl and alfentanil (Short et al. 1992). Fentanyl was 134 selected to sedate the dogs in our study because of its cardiovascular stability and common use 135 for pre-medication in sick clinical cases. 136

137

INJECTABLE INDUCTION AGENTS 138

Small animal practices in North America and Europe typically induce unconsciousness 139 with injectable anesthetic agents intravenously. Propofol and alfaxalone are two common and 140 readily available injectable induction agents, which are licensed for use in dogs and cats in South 141

Africa, Australia, Canada, the United States, and Europe. 142

143

ALFAXALONE 144

The first described anesthetic use of steroids, especially pregnane and androstane, date 145 back to 1941 (Selye 1941). Over time, many different compounds have been developed and 146 studied for potential clinical application. Among these, AlthesinTM the human product or 147

SaffanTM the veterinary product, which contained 9 mg mL-1 alfaxalone and 3mg mL-1 148 alfadolone, formulated in Cremophor EL, had been extensively used in both humans and animals 149 clinically. Alfaxalone is the active anesthetic of this combination. Cremophor EL was used as a 150 non-ionic detergent to dissolve alfaxalone and combined with an additional steroid, alfadolone 151 acetate, to further enhance the solubility of alfaxalone in the Cremophor EL vehicle. However 152

Cremophor EL typically caused histamine release that resulted in allergic reactions resembling 153 anaphylactic shock, including hypotension, bronchospasm, cardiovascular collapse, urticaria and 154 erythema in humans and dogs, and also caused hyperaemia and edema in the pinnae and 155 forepaws in 70% of cats (Child et al. 1971; Dodman 1980). These adverse effects, most 156 importantly hypotension and even death in people, eventually lead to withdrawal of these 157 products from the market in most countries. 158

Alfaxalone, is now formulated with 2-hydroxypropyl-beta-cyclodextrin and does not 159 cause histamine-mediated allergic reactions. It was introduced to the Canadian market in 2008 160

7 and is licensed as an intravenous induction agent in dogs and cats at a dose of 2-3 mg kg-1 in 161 dogs and 5 mg kg-1 in cats. It is not licensed for intramuscular (IM) use in Canada, as it is in 162

Australia and New Zealand. 163

164

Physical and chemical properties 165

Alfaxalone is a clear, colorless, aqueous solution in a multi-use clear 10 mL vial. Since 166 the vial does not contain preservative, any remaining drug should be discarded within 24 hours 167 after opening and first use. However the company has suggested that alfaxalone may be stored at 168

4 °C for up to 7 days after it is opened (Jurox Pty Ltd, Australia). Nevertheless, Strachan et al, 169

2008, demonstrated exponential growth of Escherichia coli 24 hours after inoculation of two 170 common environmental bacteria, Staphylococcus aureus and Escherichia coli, with alfaxalone on 171 agar plate at 37°C (Strachan et al. 2008). Hence, caution should be exercised when keeping the 172 opened alfaxalone vial for longer than the company recommends. 173

The alfaxalone formulation is composed of 1% alfaxalone, less than 10% 174

2-hydroxypropyl-beta-cyclodextrin, less than 1% non-hazardous ingredient and water to a 100% 175 solution. Alfaxalone has a specific gravity of 1.02-1.03. a pH of 6.5-7.0, and is stable for 36 176 months after manufacturing. 177

The chemical structure is 3 alpha-hydroxy, 5 alpha-pregnane 11,20 dione. From 178 persistent screening and testing of analogues over decades, scientists have discovered that 179 oxygenation at each end of the steroid molecule is necessary for anesthetic activity (Sear 1996). 180

However, substitutions at the other sites decreases anesthetic potency (Kumar et al. 1993). Also, 181 the 3 alpha-hydroxyl shows a higher potency over the 3 beta-hydroxyl group (Sear 1996). 182

183

Pharmacology 184

Central nervous system effects 185

The exact mechanism of action of injectable anesthetics to induce general anesthesia 186 remains under great debate. Many theories have been developed and examined such as an 187 interaction with membrane lipid bilayers or membrane-bound proteins. However, controversial 188 aspects of membrane lipid bilayer and voltage-gated ion channel interactions have resulted in the 189 general conclusion that ligand-gated ion channels are the major sites of action (Pleuvry 2004). 190

Among numerous ligand-gated ion channels, post-synaptic gamma-aminobutyric acid type A 191

(GABAA) receptor is the common site of action shared by many anesthetics including 192 neurosteroids (Akk et al. 2007). Potentiation of both phasic (transient) and tonic (persistent) 193 transmission by neurosteroids to decrease neuronal excitability has been suggested (Akk et al. 194

2007). Neurosteroids augment phasic transmission by prolonging the inhibitory current without a 195 change in peak amplitude (Harrison et al. 1987). Moreover, through either direct activation or 196 potentiation of GABA effects on extra-synaptic site receptors, neurosteroids increase tonic 197 transmission (Stell et al. 2003; Shu et al. 2004). 198

AlthesinTM has been shown to decrease cerebral metabolic rate and lower intracranial 199 pressure in spontaneously breathing people with normal or high intracranial pressure, either 200 induced by ketamine or trauma (Sari et al. 1976; Ekhart et al. 1979; Bullock et al. 1986). Also, it 201 has been found that the decrease in intracranial pressure is reversible without a dangerous 202 rebound in pressure in humans (Zattoni et al. 1980). These benefits, with minimal negative 203 cardiovascular effects, have resulted in AlthesinTM being recommended over barbiturates in 204 patients with intracranial hypertension (Bullock et al. 1986). 205

206

A reduction in intracranial pressure following administration of AlthesinTM was also 207 observed in healthy cats (Baldy-Moulinier & Besset-Lehmann 1975). However, in this study the 208 cerebral vasculature remained responsive to arterial carbon dioxide levels and hypercapnia 209 diminished the reduction in intracranial pressure from AlthesinTM (Baldy-Moulinier & 210

Besset-Lehmann 1975; Baldy-Moulinier et al. 1975). Unlike the reports in people and cats, 211

TIVA with AlthesinTM did not alter cerebral blood flow or intracranial pressure in healthy dogs 212

(Cohen et al. 1973). To the author’s knowledge, no scientific data regarding the effects of 213 alfaxalone on the cerebral vasculature have been published for dogs and cats. However, since the 214 active ingredient in AlthesinTM is alfaxalone, it is likely that the described effects on the central 215 nervous system and neurologic outcomes apply. 216

217

Cardiovascular effects (see Table 1) 218

Alfaxalone causes a dose-dependent minimal to moderate reduction in systemic vascular 219 resistance (SVR) and mean arterial pressure (MAP), and minimal to mild increases in heart rate 220

(HR) and CI, in both sedated and unsedated healthy dogs, at clinically relevant induction doses 221

(l.5-4.15 mg kg-1, IV) (Ambros et al. 2008; Muir et al. 2008; Rodriguez et al. 2012; Amengual et 222 al. 2013; Maney et al. 2013). Nevertheless, supra-clinical doses (20 mg kg-1) can result in 223 moderate decreases in SVR (2657 to 1781 dynes sec-1 cm-5; 67%), MAP (123 to 65 mmHg; 52%), 224 and CI (246 to 190 mL kg-1 minute-1; 77%), despite minimal effects on HR (unchanged at 225

128-131 beats minute-1) (Muir et al. 2008). 226

In general, alfaxalone causes a similar decrease in MAP when compared to propofol for 227 anesthetic induction, although MAP remains within the normal range (Ambros et al. 2008; 228

Amengual et al. 2013; Maney et al. 2013). However, in one study the results trended toward 229

10 higher HR and CI and lower SVR with alfaxalone than propofol despite of no statistical 230 significant difference in acepromazine and hydromorphone sedated dogs (Ambros et al. 2008). 231

Increases in HR following administration of alfaxalone have been reported in dogs sedated with 232 fentanyl (Okushima et al. 2015), acepromazine with meperidine (Amengual et al. 2013), and 233 unsedated dogs (Maney et al. 2013). These results indicate that the baroreceptor reflex might be 234 better preserved by alfaxalone than propofol. However, further studies are required to assess the 235 repeatability of this result and potential significance for cardiovascular effects. 236

Etomidate has been shown to cause minimal cardiovascular effects including in 237 hypovolemic dogs (Pascoe et al. 1992). Alfaxalone resulted in similar cardiovascular effects after 238 induction to those of etomidate in unsedated healthy research dogs (Rodriguez et al. 2012). This 239 included an increase in HR and CI and decrease in SVR caused by alfaxalone that was 240 significantly different from baseline. The ability of this study to demonstrate significant changes 241 following alfaloxone administration is possibly due to the use of unsedated healthy research dogs 242 and the use of a higher dose than in other studies where alfaxalone was compared to propofol 243 without significant differences being found (Rodriguez et al. 2012). 244

In compromised dogs (American Society of Anesthesiologists classification system [ASA] 245 three to five; Three: severe systemic disease, Four: severe systemic disease constantly threat to 246 life, Five: moribund patients not expected to survive 24 hours with or without operation), 247 induction with alfaxalone resulted in similar MAP and higher HR than dogs induced with a 248 combination of fentanyl-diazepam, a commonly used safe combination for this type of patients 249

(Psatha et al. 2011). Therefore alfaxalone is a suitable alternative to fentanyl-diazepam. 250

Overall, when considering the cardiovascular effects induction with, alfaxalone is at least 251 equivalent to propofol, etomidate and diazepam/fentanyl in healthy dogs. However, more studies 252

11 are needed in clinically compromised dogs to completely assess the cardio-pulmonary profiles, 253 as in healthy dogs the cardio-pulmonary parameters remained within acceptable range despite 254 minor differences between induction agents. 255

256

Respiratory effects 257

Alfaxalone has been shown to induce dose-dependent respiratory depression ranging 258 from a reduction in respiratory rate and minute volume to complete apnea at various doses 259 ranging from 2 to 40 mg kg-1, IV, in dogs 8 months to 10 years old (Muir et al. 2008; Keates & 260

Whittem 2012). Nevertheless, the tidal volume has been shown to remain stable with as high as 6 261 and 20 mg kg-1 bolus injections, IV (Muir et al. 2008). With use of a smaller clinical dose of 2 262 mg kg-1, IV, a shorter and lower degree of apnea was noted in adult dogs than was observed at 263 doses of 6 and 20 mg kg-1 (Muir et al. 2008). In dogs under 12 weeks of age, alfaxalone 264 administration at 1.7 ± 0.3 mg kg-1, IV, at the rate of 2 mg second-1 resulted in apnea in 1 of the 265

25 dogs (O'Hagan et al. 2012). These results demonstrate that alfaxalone has a wide margin of 266 respiratory safety in healthy dogs of various ages, especially at clinically relevant doses. 267

Various studies have compared the respiratory effects between propofol and alfaxalone. 268

Doses of 1, 2, 5, 10 and 20 times the clinical doses of propofol (6.5 mg kg-1, IV) and alfaxalone 269

(2 mg kg-1, IV) were compared for bolus injection inducing apnea in 6 dogs and alfaxalone did 270 not cause apnea in all dogs until 10 times the clinical dose was administered, whereas propofol 271 caused apnea in 2 of 6 dogs at 5 times the clinical dose (Keates & Whittem 2012). However, 272 other researchers did not report differences in respiratory function between propofol and 273 alfaxalone (Ambros et al. 2008; Amengual et al. 2013; Maney et al. 2013). For example, 274 acepromazine 0.03 mg kg-1 and meperidine 3 mg kg-1 given 30 minutes prior to administration of 275

3 mg kg-1 propofol or 1.5 mg kg-1 alfaxalone, as rapid bolus injections, IV, in 6 sedated dogs 276 breathing spontaneously (FiO2=1) resulted in similar numbers of apneic dogs and similar degree 277 of increase in arterial carbon dioxide tensions between the groups (Amengual et al. 2013). 278

Unsedated dogs breathing spontaneously (FiO2=0.21) given propofol or alfaxalone to effect until 279 endotracheal intubation was achieved did not demonstrate apnea (Maney et al. 2013). 280

Furthermore, there were no differences between propofol or alfaxalone groups for blood pH, 281 base excess, and end-tidal and arterial carbon dioxide tensions, but the alveolar-arterial oxygen 282 tension gradient increased as a result of a decrease in arterial oxygen tension in both group 283

(Maney et al. 2013). In another study, providing an FiO2=1 in sedated dogs breathing 284 spontaneously, no differences in percentage of apnea (1 out of 6), shunt fraction or alveolar 285 dead-space were noted between propofol and alfaxalone (Ambros et al. 2008). 286

When compared to etomidate, alfaxalone demonstrates a similar degree of limited 287 respiratory depression in terms of the end-tidal and arterial carbon dioxide tensions and arterial 288 oxygen tension in healthy unsedated dogs (Rodriguez et al. 2012). A similar post-induction 289 respiratory rate is shown between diazepam/fentanyl and alfaxalone in clinically compromised 290 dogs (ASA 3-5) (Psatha et al. 2011). 291

In conclusion, alfaxalone exhibits similar, or possibly reduced, respiratory depression in 292 healthy dogs whether sedated or not compared to propofol and etomidate. Alfaxalone, even in 293 compromised patients, may not compromise respiratory function significantly. Additional 294 research in compromised canine patients is warranted in clinical cases to fully define the 295 respiratory effects when combined with other anesthetic and analgesic agents. 296

297

298

Induction dosage and quality (see Table 2) 299

The induction IV dose of alfaxalone in unsedated and sedated healthy dogs ranges from 2 300 to 4.15 mg kg-1 and 0.5 to 1.9 mg kg-1, respectively. Even without premedication, the quality of 301 induction with alfaxalone is repored as good to smooth (Muir et al. 2008; Rodriguez et al. 2012; 302

Maney et al. 2013). The quality of induction has been found to be comparable to propofol 303

(Ambros et al. 2008; Suarez et al. 2012; Amengual et al. 2013; Maney et al. 2013) and etomidate 304

(Rodriguez et al. 2012). Reported side effects in dogs include excitement, paddling or muscle 305 twitching (Maddern et al. 2010; Amengual et al. 2013; Maney et al. 2013). Pre-medication is 306 recommended by the manufacturer (Jurox Pty Ltd, Australia) within the product monograph to 307 prevent excitement and rough recoveries in dogs. Because alfaxalone is rapidly re-distributed, 308 the company recommends an initial dose of 2-3 mg kg-1 in dogs to prevent arousal during the 309 induction phase and/or transfer to maintenance phase. Clinically, with appropriate sedation, these 310 higher doses may not be required, especially if a co-induction agent is also administered. 311

312

PROPOFOL 313

Physical and chemical properties 314

The development of propofol dates back to the 1970s. The chemical structure of propofol 315 is 2,6-di-isopropylphenol, which is in oil form and insoluble in aqueous solvents at room 316 temperature. It was formulated with Cremorphor EL initially, however, the anaphylactoid side 317 effects and pain during injection of Cremorphor EL led to the development of a new formulation 318 containing 1% propofol, 10% soybean oil, 2.25% glycerol, 1.2% purified egg phosphatide, which 319 results in a preparation with a pH of around 7, and a milky white appearance (Short & Bufalari 320

1999). It is not light sensitive and is stable at room temperature (Miller 2005). However, there is 321

14 no preservative in the formulation, hence, all remaining drug should be discarded 6 hours after 322 opening and first use (Tranquilli et al. 2007). 323

324

Pharmacology 325

Central nervous system effects 326

Propofol primarily acts on GABAA receptors, as well as alpha2-adrenoreceptors, 327

N-methyl-D-aspartate (NMDA) receptors, and glycine receptors (Miller 2005). Recognized CNS 328 effects of propofol in dogs include a decrease in cerebral oxygen consumption and cerebral blood 329 flow, a decrease in intracranial pressure, and preservation of autoregulation of arterial carbon 330 dioxide tensions until high doses are administered (Artru et al. 1992) or conditions of hypoxia 331 are present (Haberer et al. 1993). 332

333

Cardiovascular effects 334

Like alfaxalone, propofol has negative cardiovascular effects in animals and people. 335

When compared to etomidate for induction, propofol causes lower MAP despite a higher HR 336

(Sams et al. 2008). Dose-dependent suppression of preload, contractility and lusitropy have been 337 shown with propofol (Puttick et al. 1992). Despite this, no direct suppression of cardiac 338 contractility is reported in dogs until supraclinical plasma concentrations (more than 7µg mL-1) 339 are achieved (0.4 mg kg-1 min-1), which suggests indirect myocardial depression through a 340 central effect (Ismail et al. 1992; Belo et al. 1994; Kawakubo et al. 1999). Also, there is evidence 341 suggesting that the current adjuvants (intrafat) within propofol cause vasoconstriction at 342 relatively low concentration but vasodilation at relatively high concentration in isolated dog 343 arteries (Nakamura et al. 1992). The underlying vasodilation mechanism of the adjuvants has 344

15 been related to nitric oxide pathway stimulation (Doursout et al. 2002) and either direct 345

(Goodchild & Serrao 1989; Nakamura et al. 1992) or indirect effects (Robinson et al. 1997) of 346 propofol. These effects, in combination with the fact that the baroreceptor reflex sensitivity is 347 reset by propofol (Whitwam et al. 2000), result in a decrease in arterial blood pressure when 348 propofol is used for anesthetic induction. Moreover, depression of the global cardiovascular 349 effects from propofol induction have been reported in acutely hypovolemic dogs and it was not 350 recommended for these cases (Ilkiw et al. 1992). Hence, titrating propofol to effect to the 351 anesthetic depth required is important to minimize these negative cardiovascular effects. 352

353

Respiratory effects 354

Propofol has well documented respiratory depression and commonly results in 355 post-induction apnea and cyanosis (Muir & Gadawski 1998). The apnea is both rate- and 356 dose-dependent (Muir & Gadawski 1998). However, there is evidence to support an association 357 between both rapid (Muir & Gadawski 1998) or slow (Murison 2001) injection with 358 post-induction apnea. Hypoventilation, or elevated arterial carbon dioxide (PaCO2), is also 359 common after propofol injection (Ambros et al. 2008). As outlined above, comparisons between 360 propofol and alfaxalone have varying results. Propofol has been shown to have more respiratory 361 depressive effects compared to alfaxalone (Keates & Whittem 2012), or similar mild respiratory 362 effects (Ambros et al. 2008; Amengual et al. 2013; Maney et al. 2013). When compared to 363 etomidate for induction, propofol causes a higher degree of respiratory depression that results in 364 higher PaCO2 and lower PaO2 (Sams et al. 2008), but similar effects to thiopental 365

(Redondo-Garcia et al. 1999). 366

367

Induction dosage and quality 368

The induction dose of propofol in dogs ranges from 2.6 to 5.5 mg kg-1 (Plumb 2011). The 369 appropriate induction dose depends largely on the level of sedation in the animals achieved with 370 premedication, the alertness and health status of the animal, and the injection rate (Amengual et 371 al. 2013). At low doses, 0.5-1 mg kg-1, IV, propofol has sedative effects (Yoon et al. 2002). 372

Even though the induction quality of propofol in dogs is generally reported as satisfactory, 373 excitement, dystonia, involuntary muscle contractions, paddling, opisthotonus and 374 hyperextension have been reported (Davies & Hall 1991; Robertson et al. 1992; Smedile et al. 375

1996; Mitek et al. 2013) and clinically significant (Hall & Chambers 1987; Duke 1995). A recent 376 retrospective study that excluded those cases with inadequate analgesia and/or depth of 377 anesthesia by administration of an IV bolus of fentanyl or propofol showed a lower incidence of 378 adverse neurological effects (6 out of 492;1.2%) (Cattai et al. 2015). These side effects are 379 postulated to be due to antagonism of inhibitory glycine receptors at the subcortical level and 380 imbalance between inhibitory dopamine receptors and excitatory cholinergic receptors in the 381 basal ganglia especially during rapid change of the concentration in the brain (San-juan et al. 382

2010). In humans, electroencephalography has been performed during and after the abnormal 383 activity and showed a lack of typical seizure patterns in most patients suggesting that the 384 observed effects are seizure-like phenomenon (SLP) instead of a true seizure (San-juan et al. 385

2010). Appropriate sedation is thought to reduce the incidence of SLP in dogs and cats as was 386 noted in a personal observation (Duke 1995) But in a retrospective study comparing SLP activity 387 between dogs premedicated with methadone with or without acepromazine and 388 non-premedicated dogs, the reported SLP prevalence was higher in premedicated dogs (1 out of 389

58; 1.72%) compared to non-premedicated dogs (5 out of 432; 1.15 %) (Cattai et al. 2015). 390

Therefore there is currently insufficient evidence to support the clinical impression that 391 premedication may reduce SLP activity in dogs undergoing propofol induction and/or TIVA. 392

Further studies are needed regarding the effects of different premedications on the incidence of 393 adverse effects associated with propofol administration. 394

395

THE USE OF CO-INDUCTION AGENTS 396

Advantages 397

The main reasons for using a co-induction agent with an injectable anesthetic agent are to 398 smooth the overall induction process enabling endotracheal intubation without swallowing or 399 coughing, minimize the negative side effects of the primary induction agent (cardiovascular and 400 respiratory), minimize the dose of injectable drug administered and thereby lower the overall 401 cost, and promote a smoother transfer to the maintenance phase of anesthesia. The main 402 advantages of using different types of drugs together during the induction of anesthesia in 403 cardiovascularly compromised or critical patients are the lowered dose of the primary anesthetic 404 induction agent, ease of endotracheal intubation and enhancement of both cardiorespiratory 405 stability and muscle relaxation (Whitwam 1995). Common co-induction agents used in dogs are 406 lidocaine, diazepam or midazolam and ketamine. Fentanyl, 2 µg kg-1, IV, over 30 seconds, 2 407 minutes before propofol has been used in one canine study as a co-induction drug, after 408 acepromazine and morphine sedation, and showed a 18% dose reduction (Covey-Crump & 409

Murison 2008). However, opioids are more commonly used in the premedication and 410 maintenance phases of anesthesia than as co-induction agents in veterinary medicine to date. 411

Results vary in the literature depending on the agent, dosage, induction technique (CRI or 412 boluses), order of administration (before or after the induction agent), speed of injection of the 413

18 co-induction, and the primary induction agent used. For example, when used as a co-induction 414 with propofol, an 18% propofol dose reduction and improved induction quality was 415 demonstrated with fentanyl 2 µg kg-1, IV, but not midazolam 0.25 mg kg-1, IV, (Covey-Crump & 416

Murrison 2008); whereas other investigations have demonstrated a dose reduction with diazepam 417 or midazolam (0.2 or 0.5 mg kg-1) used with propofol as the primary induction agent (Robinson 418

& Borer-Weir 2013; Sanchez et al. 2013). 419

420

Disadvantages 421

The disadvantages of using a co-induction agent are potential excitement, the additional 422 step of administering the co-induction agent, added cost of the co-induction agent, the necessary 423 individual drug pharmacological knowledge, and potential drug interactions. Patient excitement 424 is especially problematic for benzodiazepines, and may result in a higher dose of the induction 425 agent. Proper use of co-induction agents requires training, skill and appropriate depth assessment 426 to prevent excessive anesthetic depth and overdose. Proper monitoring with close assessment is 427 key for patients undergoing co-induction. 428

429

Drugs commonly used as co-induction agents 430

Benzodiazepines 431

Benzodiazepines act primarily on the GABAA enhancing the binding between receptor 432 and neurotransmitter and hence increasing the frequency of receptor opening (Tranquilli et al. 433

2007). However, since benzodiazepines do not possess intrinsic agonist activity, there is a ceiling 434 to the sedative effect achieved even when a high dose is given (Saari et al. 2011). 435

436

In people, benzodiazepines cause greater sedation and even unconsciousness, however, 437 on their own, benzodiazepines are not profound sedatives in dogs and cats. Moreover, excitement 438 may appear due to “disinhibition” when given as a sole agent in healthy dogs (Haskins et al. 439

1986; Court & Greenblatt 1992). In people, the benzodiazepine may be given 30 minutes prior to 440 the primary induction agent (Short et al. 1992). Due to these differences, benzodiazepines have 441 been primarily investigated as co-induction agents in veterinary medicine with timing close to 442 the primary induction agent (immediately before or after). 443

Current veterinary literature shows conflicting results in dogs regarding the dose 444 reduction when benzodiazepines are used as co-induction agents with propofol. No reports are 445 available for the use of benzodiazepines with alfaxalone in dogs but co-induction with 446 midazolam showed dose reduction of alfaxalone in goats (Dzikiti et al. 2014). In earlier studies, a 447 diazepam, 0.4 mg kg-1, IV bolus, given 45 seconds prior to propofol titrated to effect, provided a 448

36% dose reduction of propofol (Ko et al. 2006) but this dose reduction was not seen when a 449 dose of diazepam of 0.2 or 0.25 mg kg-1, IV, bolus was given 45 seconds or two minutes prior to 450 propofol (Ko et al. 2006; Braun et al. 2007). In contrast, when given after 1 mg kg-1 propofol, IV 451 over 15-45 seconds, diazepam showed no dose reduction from 0.2 to 0.5 mg kg-1, IV bolus 452

(Robinson & Borer-Weir 2013). The different results in these investigations could be attributed 453 to sedation level of the dogs, dose of diazepam, order of administration (before or after the initial 454 bolus of propofol), speed of diazepam administration (bolus-slow injection over 30 seconds), and 455 initial dose and rate of propofol administration. 456

The canine literature using midazolam as a co-induction agent also demonstrates 457 conflicting results, likely related to those factors listed above. However, the results with 458 midazolam are typically favorable. Midazolam, 0.25 mg kg-1, given IV over 30 seconds two 459

20 minutes prior to propofol induction, did not cause dose reduction but resulted in excitement in 21 460 of 22 (95%) dogs, despite prior IM sedation with acepromazine (0.025 mg kg-1) and morphine 461

(0.25 mg kg-1) 30 minutes before induction (Covey-Crump & Murison 2008). Administration of 462 midazolam, 0.25 mg kg-1, given IV over 1 minute, 30 seconds prior to 1 mg kg-1 propofol, 463 demonstrated a 47 % dose reduction with excitement in 5 of 11 (45%) dogs previously sedated 464

IM with acepromazine (0.02 mg kg-1) and morphine (0.4 mg kg-1) 30 minutes before induction 465

(Sanchez et al. 2013). In another study, this same dose of midazolam (midazolam, 0.25 mg kg-1, 466

IV) given over 15 seconds, immediately prior to propofol, showed a 18% dose reduction and 467 excitement in 5 of 9 (55%) dogs that were sedated IM with acepromazine (0.025 mg kg-1) and 468 morphine (0.25 mg kg-1) 30 minutes before induction (Hopkins et al. 2013). The lower incidence 469 of excitement in these latter studies is likely attributed to a shorter time delay between the 470 administration of the midazolam and propofol. Reports also indicate that when midazolam is 471 given after propofol, at 1 mg kg-1, IV, a consistent significant dose reduction (38-66%) and lower 472 excitement percentage (12-18%) is seen (Robinson & Borer-Weir 2013; Sanchez et al. 2013). 473

Considering the results of these studies, it appears that when choosing diazepam as a 474 co-induction agent, a higher dose (0.4 mg kg-1, IV) administered before propofol is most likely to 475 cause a dose reduction of propofol. These findings coincide with the slower onset and lower 476 potency characteristics of diazepam when compared to midazolam. When using midazolam as a 477 co-induction agent, the timing between midazolam and propofol administration and speed of 478 midazolam administration are the two main factors related to the demonstration of excitement 479 and dose reduction with propofol. Nevertheless, midazolam still demonstrates the most 480 promising performance when used as a co-induction agent with propofol and potentially with 481 other injectable anesthetics. Due to these more consistent findings in the literature, midazolam 482

21 was chosen in our studies. 483

The actual mechanism of benzodiazepines to promote dose reduction and allow for a 484 smoother induction is still unclear. Synergism and additive effects with the primary induction 485 agent are most likely. In vitro research shows that benzodiazepines act like potentiators through 486 changing gating equilibrium of GABAA receptors to different kinds of allosteric agonists (Li et al. 487

2013). 488

Midazolam is a benzodiazepine that contains a fused imidazole ring, which has a 489 pH-dependent ring-opening phenomenon. The formulation is in aqueous form and is light 490 sensitive. It is not irritating and is well absorbed via intramuscular injection. Midazolam is 491 primarily metabolized in the liver and excreted through the kidneys. The lipophilicity of 492 midazolam is higher than diazepam (Tranquilli et al. 2007) and the affinity to the GABAA 493 receptor is double (Mohler & Okada 1977). Also, midazolam has shown higher potency than 494 diazepam in humans (Reves et al. 1978; Buhrer et al. 1990) and dogs regarding to the elevation 495 of the threshold of lidocaine-induced seizure in dogs (Horikawa et al. 1990). However, because 496 of lower intrinsic potency of benzodiazepines in dogs compared to humans and similar anesthetic 497 dose used clinically, further study is needed regarding to anesthetic potency between midazolam 498 and diazepam in dogs. The pharmacokinetic profile of midazolam has been reported in number 499 of studies. In conscious dogs: the volume of distribution in steady state after 0.2 mg kg-1, IV, 500 ranged from 0.68 ± 0.33 L kg-1 (Schwartz et al. 2013); the volume of distribution using area 501 method after 0.2 – 0.5 mg kg-1, IV, ranged from 1.10 ± 0.56 – 3.00 ± 0.90 L kg-1 (Court & 502

Greenblatt 1992; Schwartz et al. 2013); the clearance after 0.2 – 0.5 mg kg-1, IV, 10.1 ±1.9 – 27 503

± 3 mL min-1 kg-1 (Court & Greenblatt 1992; Schwartz et al. 2013); the elimination half life after 504

0.2 – 0.5 mg kg-1, IV, ranged from 63.3 ± 28.5 – 121 ± 6 minutes (Court & Greenblatt 1992; 505

Schwartz et al. 2013). In enflurane anesthetized dogs given midazolam 2.5 mg kg-1, IV, the 506 volume of distribution using area is 3.94 ± 0.27 L kg-1, the clearance is 28.5 ± 3.1 mL min-1 kg-1, 507 the elimination half-life is 98 ± 5 minutes (Hall et al. 1988b). 508

In dogs, clinical doses of midazolam are between 0.1 to 0.5 mg kg-1, administered by IV 509 or IM routes (Plumb 2011). The cardiovascular effects of midazolam at doses between 0.25, 1 510 and 10 mg kg-1 are minimal in dogs (Jones et al. 1979). For all doses in the aforementioned study, 511

HR increased by 10 to 20 % and CO increased by 10 to 12 % following midazolam 512 administration (Jones et al. 1979). However, MAP decreased by 10 to 20 % and cardiac 513 contractility decreased by 13 to 16 % at the 1 and 10 mg kg-1 doses (Jones et al. 1979). Moreover, 514

SVR decreased when 1 and 10 mg kg-1 doses were given (Jones et al. 1979). Neither doses 515 decreased stroke volume (SV) (Jones et al. 1979). Despite benzodiazepines deemed as rarely 516 causing respiratory depression, some studies have shown respiratory depression, as evidenced by 517 a decrease in tidal volume and oxygen saturation, and an increase in end-tidal CO2 with clinical 518 doses in anesthetized dogs (Heniff et al. 1997). Midazolam exhibits dose-dependent isoflurane 519 and enflurane MAC reduction in dogs with maximum about 30% and 60-70 % respectively (Hall 520 et al. 1988a; Seddighi et al. 2011). 521

522

Lidocaine 523

Lidocaine is one of the most extensively used local anesthetics. When used in regional 524 anesthesia, it provides intermediate duration and fast onset. Intravenous infusions of lidocaine 525 also provide anesthetic and analgesic effects (Tranquilli et al. 2007). 526

Co-induction with lidocaine, IV, in humans has been shown to decrease propofol 527 injection pain (Borazan et al. 2012) in addition to suppression of coughing (Yukioka et al. 1985) 528

23 and sympathetic responses (Mark et al. 1987) associated with endotracheal intubation. Despite 529 these benefits, dose sparing effect has not been shown in either people (Tan & Hwang 2003) or 530 dogs with lidocaine co-induction administration with propofol (Braun et al. 2007; Jolliffe et al. 531

2007). 532

Furthermore, co-induction with lidocaine was not beneficial in decreasing the incidence 533 of coughing or attenuating sympathetic responses in dogs induced with propofol (Jolliffe et al. 534

2007). No investigations have been performed of lidocaine co-induction with alfaxalone. More 535 research is warranted to clearly outline the advantages and disadvantages of lidocaine 536 co-administration with both propofol and alfaxalone in dogs. 537

538

Ketamine 539

Ketamine is a dissociative anesthetic that is used routinely in veterinary medicine with 540 diazepam or midazolam for anesthetic induction without propofol or alfaxalone. However, it has 541 also been used at lowered doses (1-2 mg kg-1) in combination with propofol at induction. In this 542 context, it is being used as a co-induction agent (Lerche et al. 2000; Mair et al. 2009; 543

Martinez-Taboada & Leece 2014). 544

Ketamine has unique pharmacology. The main action of ketamine on the central nervous 545 system is to interrupt the connection of cerebral cortex and thalamus and hence causes 546 characteristic anesthesia status-catalepsy (Miller 2010). In addition to the dose reduction and 547 analgesic effects, the rationale of using ketamine as a co-induction agent is the sympathomimetic 548 effect (Miller 2010) that might counteract the cardiovascular depressive effects of propofol or 549 potentially alfaxalone that occur, especially at higher doses. 550

However, ketamine also produces negative inotropic effects (Diaz et al. 1976) which are 551

24 often masked by the direct sympathetic stimulation in healthy animals. Hence, ketamine should 552 still be used with caution in cardiovascular compromised patients. The use of ketamine, 0.25-0.5 553 mg kg-1, IV, given one minute before propofol to effect, did not demonstrate a dose reduction or 554 improved cardiovascular function in dogs (Mair et al. 2009). However, when ketamine was 555 mixed with propofol to form 9 mg mL-1 of each drug, the total propofol dose (0.2 ± 0.1 mg kg-1) 556 was significantly lower than for propofol alone (0.4 ± 0.1 mg kg-1). This was accompanied by a 557 significant increase in MAP and HR (Martinez-Taboada & Leece 2014). In addition, ketamine, at 558

2 mg kg-1, IV, given after propofol, at 2 mg kg-1, IV, resulted in a higher HR than propofol alone, 559 at 4 mg kg-1, IV, despite similar MAP (Lerche et al. 2000). Hence, ketamine may counteract the 560 negative cardiovascular effects of propofol induction with or without dose reduction in healthy 561 dogs. 562

In summary, co-induction agents have the potential benefit of lowering the dose of the 563 primary induction agent, thereby reducing any of the negative cardio-pulmonary effects. Yet, 564 such cardio-pulmonary and dose reducing benefits are not always observed and the optimal 565 protocol is not clearly defined in the research to date in dogs. No investigations are available in 566 which co-induction agents are used in concert with alfaxalone in dogs other than a case series 567 study investigating the induction effects of the alfaxalone and midazolam (Seo et al. 2015). 568

569

TOTAL INTRAVENOUS ANESTHESIA (TIVA) 570

History of TIVA 571

After induction from consciousness to unconsciousness with an injectable anesthetic 572 intravenously, veterinary patients are typically maintained with inhalant anesthetics during the 573 surgical or diagnostic procedure. However, in some instances the inhalant anesthetic may pose a 574

25 challenge or have physiologic disadvantages warranting maintenance of anesthesia with an 575 injectable anesthetic, termed total intravenous anesthesia (TIVA). Of the injectable anesthetics 576 available for veterinary use, propofol and alfaxalone possess ideal pharmacological profiles for 577

TIVA. In fact, propofol has been used in human anesthesia as a maintenance agent since the late 578

80’s (Shafer et al. 1988). 579

580

TIVA compared to inhalant anesthesia 581

The advantages of maintenance with inhalants are the predictability, rapid adjustment 582 with only minimal metabolism and excretion (Waelbers 2009). However, specialized vaporizers 583 and equipment are required for precise and safe delivery of inhalants. Moreover, pollution into 584 the surrounding workspace space presents serious health (Ornoy 2012) and environmental 585

(Goyal & Kapoor 2011) hazards with risks of chronic human exposure and accumulation of the 586 exhausted inhalant in the atmosphere and ozone layer being identified. 587

With propofol and alfaxalone TIVA, occupational health and environmental concerns are 588 alleviated and patient drug metabolism and excretion are less of a concern than for other 589 injectable agents (Hatschbach et al. 2008). Other positive factors have been demonstrated in 590 humans including lower levels of postoperative nausea and less emergence excitement (Lerman 591

& Johr 2009). There is also evidence in dogs that inhalant maintenance is associated with more 592 hypotension (Iizuka et al. 2013a) and requires more frequent use of vasopressors (Caines 2013), 593 whereas propofol has been shown to better preserve MAP and aortic compliance (Deryck et al. 594

1996). The main disadvantage and concern with TIVA is the cost, especially when the duration 595 of anesthesia required exceeds one hour (Short & Bufalari 1999). However, with proper 596 co-administration of analgesics and sedatives, successful dose and cost reduction is achievable 597

(Waelbers 2009). 598

Use of alfaxalone as a total intravenous agent 599

Pharmacokinetics of alfaxalone 600

The pharmacokinetics of alfaxalone have been investigated in research dogs . The 601 volume of distribution, elimination half-life, and plasma clearance of 2 mg kg-1, IV bolus 602 injection, are 2.4 ± 0.9 L kg-1, 24.0 ± 1.9 minutes, and 59.4 ± 12.9 mL minute kg-1, respectively; 603 and for 10 mg kg-1 are 2.9 ± 0.4 L kg-1, 37.4 ± 1.6 minutes and 52.9 ± 12.8 mL minute kg-1, 604 respectively (Ferre et al. 2006). 605

Similar short times to endotracheal intubation (less than a minute) have been shown in 606 research dogs after a single bolus (Ferre et al. 2006; Muir et al. 2008). The rapid onset and 607 recovery of alfaxalone make it suitable for TIVA in dogs. 608

609

Cardiovascular and respiratory effects of alfaxalone TIVA 610

Alfaxalone causes minimal cardiovascular changes when used as a maintenance agent. A 611 dose of 0.07 mg kg-1 min-1 for 120 minutes in dogs maintained anesthetized without surgical 612 stimulation did not significantly affect MAP, pulmonary artery pressures, right atrium pressure, 613 pulmonary artery wedge pressure, HR, CI, SVI, and SVR compared to before induction (Ambros 614 et al. 2008). Minimal to mild cardiovascular depression has been shown in two clinical studies 615 using alfaxalone doses of 0.08-0.11 mg kg-1 min-1, as a maintenance agent for normal healthy 616 dogs during ovariohysterectomies (Suarez et al. 2012; Herbert et al. 2013). 617

Alfaxalone TIVA without surgical stimulation causes moderate respiratory depression 618

(increased PaCO2), despite acceptable PaO2 values (FiO2=1) (Ambros et al. 2008). With surgical 619 stimulation (ovariohysterectomy), higher dose infusion of alfaxalone causes mild respiratory 620

27 depression (Suarez et al. 2012). 621

Use of propofol as a total intravenous agent 622

Pharmacokinetics of propofol TIVA 623

The pharmacokinetics of propofol have been determined in several studies and results 624 varied with different premedications, concurrent anesthetics, age and breed: the volume of 625 distiribution, elimination half-life and clearance ranged from 2.4 - 9.7 L kg-1, 14 - 486 minutes 626 and 34 -115 ml minute-1 kg-1 (Cockshott et al. 1992; Nolan & Reid 1993; Nolan et al. 1993; Reid 627

& Nolan 1993; Zoran et al. 1993; Hall et al. 1994; Mandsager et al. 1995; Reid & Nolan 1996). 628

A single bolus of IV propofol, results in rapid uptake by the central nervous system and hence 629 fast onset. This is followed by a rapid decrease in the plasma concentration due to rapid 630 redistribution and metabolism (Short & Bufalari 1999). The volume of distribution for propofol 631 is large. Hence, even though clearance and clinical recovery times are comparable to alfaxalone, 632 propofol could reside in the body longer. Propofol also exhibits unique metabolism in that the 633 metabolic clearance exceeds hepatic blood flow (Shafer 1993), suggesting extra-hepatic 634 pathways are partly responsible for metabolism. This is supported by the detection of propofol 635 metabolites during an-hepatic phases of orthotopic liver transplantation in humans (Veroli et al. 636

1992). 637

638

Cardiovascular and respiratory effects of propofol TIVA 639

Propofol causes no significant changes in HR, BP, CI, SVR, and mean pulmonary artery 640 pressure during 120 minute infusions at 0.4 mg kg-1 min-1 (Keegan & Greene 1993b). In addition, 641 both MAP and SVR are higher than during isoflurane maintenance (Keegan & Greene 1993b). In 642 a canine study with a 120 minute infusion of propofol, the cardiovascular effects were not 643

28 significantly different to alfaxalone TIVA (Ambros et al. 2008). 644

Respiratory depression is the most common side effect of propofol single doses and is 645 also observed when used as maintenance agent at the recommended doses of 0.25 mg kg-1 min-1 646

(Ambros et al. 2008). Hence, endotracheal intubation and oxygen supplementation are 647 recommended during propofol TIVA (Duke 1995). 648

649

Recovery characteristics from alfaxalone and propofol 650

Recovery quality of alfaxalone 651

The recovery quality of alfaxalone is controversial. Most studies state that the overall 652 recovery quality with alfaxalone is good to excellent (Ambros et al. 2008; Muir et al. 2008; 653

Psatha et al. 2011; Suarez et al. 2012; Herbert et al. 2013). Furthermore, recovery from 654 alfaxalone has been characterized as better than etomidate induction followed by isoflurane 655

(Rodriguez et al. 2012) and comparable with propofol induction and TIVA (Ambros et al. 2008; 656

Suarez et al. 2012) and diazepam/fentanyl induction followed by isoflurane (Psatha et al. 2011). 657

However, dogs under alfaxalone induction during MRI under sevoflurane had poorer recovery 658 scores compared to propofol induction (Jimenez et al. 2012). Also, some dogs were sensitive to 659 external stimulation (Ferre et al. 2006) or demonstrated other adverse effects such as tremors, 660 rigidity, and myoclonus at recovery (Maney et al. 2013). Hence, pre-medication as well as a 661 quiet and undisturbed environment are recommended by the company (Jurox Pty Ltd, Australia). 662

663

Recovery time with alfaxalone 664

Due to the high clearance rate of alfaxalone, recovery time is rapid. Without 665 premedication, the mean time to extubation after a single dose bolus of alfaxalone of 2 to 3, 6 666

29 and 10 mg kg-1, IV, ranges from 6.4 to 25 minutes (Ferre et al. 2006; Muir et al. 2008; Rodriguez 667 et al. 2012; Maney et al. 2013), 31.4 ± 6.9 minutes (Muir et al. 2008) and 75.1 ± 18.9 minutes 668

(Muir et al. 2008), respectively. With sedation, the mean time to extubation after CRIs of 669 alfaxalone of 80-130 minutes, with or without surgical procedures performed is 10-20 minutes 670 post-CRI (Ambros et al. 2008; Suarez et al. 2012; Herbert et al. 2013). Time to sternal and time 671 to stand after 120 minutes TIVA without surgery and 80 minutes TIVA with surgery are 14 ± 7, 672

35 ± 5, 43 ± 9 and 52 ± 10 minutes (Ambros et al. 2008) and 20 (11-22), 32 (29-40), 60 (46-61) 673 and 90 (85-107) minutes (Suarez et al. 2012). 674

675

Recovery quality of propofol 676

In general, recovery from propofol TIVA has been described as smooth and excellent 677

(Keegan & Greene 1993b; Ambros et al. 2008). However, some adverse events such as vomiting 678

(Morgan & Legge 1989), tremors, vocalization, and myoclonus (Maney et al. 2013) may occur 679 during the recovery process . 680

681

Recovery time with propofol 682

The recovery time following propofol TIVA in dogs is associated with premedication 683 agents, and breed (Robertson et al. 1992) and with total TIVA time in cats (Pascoe et al. 2006). 684

In pre-medicated and non-pre-medicated dogs, full recovery time ranged from 15 to 79.3 minutes 685 and 22 to 33 minutes, respectively (Watkins et al. 1987; Morgan & Legge 1989; Vainio 1991; 686

Robertson et al. 1992; Keegan & Greene 1993b; Thurmon et al. 1994). 687

688

Comparison between alfaxalone and propofol and for induction and maintenance 689

To date, there are only few studies comparing TIVA using alfaxalone and propofol in 690 dogs. Among those studies, time to endotracheal intubation, induction quality, and maintenance 691 quality, including the response to surgery, cardiovascular and respiratory depression, and adverse 692 events, such as muscle twitching, paddling, or myoclonus were found to be comparable (Ambros 693 et al. 2008; Suarez et al. 2012; Amengual et al. 2013; Maney et al. 2013). One study in dogs 694 demonstrated that propofol was more likely to cause apnea than alfaxalone (Keates & Whittem 695

2012). When propofol was compared to alfaxalone induction for cesarean sections in dogs, 696 puppies from the alfaxalone group demonstrated better neonatal vitality at birth however, 697 neonatal survival rates at three months were similar between the agents (Doebeli et al. 2013). 698

The comparison of cardiovascular effects of TIVA using propofol or alfaxalone during 699 mechanical ventilation in pre-medicated dogs requires further investigation as existing reports 700 allowed spontaneous breathing. 701

702

Cardiac output (CO) measurements in research 703

In any research investigating the advantages and disadvantages of anesthetic agents, 704 appropriate and accurate cardiovascular and respiratory measurements are required. Most 705 veterinary researchers use measures such as direct arterial blood pressure, HR, RR, SPO2, 706

ETCO2, arterial blood gases, temperature, and CO along with calculations of CI, SV, SVI, and 707

SVR. The measurement of CO allows for an improved assessment of overall cardiovascular 708 assessment. When using CO measurements in veterinary research, the methodology, correct 709 instrumentation and appropriate interpretation are necessary for accurate final conclusions. The 710

31 following literature supports the use of lithium dilution CO measurements and continuous pulse 711 contour CO measurements in our research. 712

713

Definition and importance of cardiac output 714

Cardiac output is “the quantity of blood pumped into the aorta each minute by the heart” 715

(Hall & Guyton 2011). Both oxygen delivery and blood pressure are important in maintaining 716 cell homeostasis and both affected by cardiac output. It has been shown in humans via numerous 717 clinical trials, reviews and meta-analyses that optimizing oxygen delivery and global blood flow 718 while under general anesthesia reduces mortality and morbidity, especially for those patients 719 who are critically ill or high-risk (Boyd et al. 1993; Gan et al. 2002; Grocott et al. 2012; Cecconi 720 et al. 2013; Salzwedel et al. 2013; Pearse et al. 2014). Moreover, it has also been shown that 721 implementation of early optimization improves outcomes (Kern & Shoemaker 2002). Hence 722 peri-operative monitoring and management of cardiac output could lead to better patient 723 outcomes. Nevertheless in veterinary medicine, it’s uncommon to measure CO due to the 724 equipment requirements and specialized personnel training needed. Instead, blood pressure is the 725 most commonly measured parameter used clinically to access cardiovascular function. However 726 if the vascular system is simplified, according to Ohm’s law, the flow through any vascular bed 727 equals the pressure difference divided by resistance. Therefore, adequate perfusion pressure does 728 not necessarily equate to adequate tissue blood flow. 729

730

Techniques of cardiac output measurement 731

The first reported CO measurement was done by A. Fick in 1870 (Fick 1870). Following 732 that, G. N. Stewart published the indicator-dilution method that used saline as indicator in 1897 733

(Stewart 1897). Over decades, different techniques for measuring CO have been developed: 734 indicator dilution methods, Fick methods, pulse contour analysis, electric impedance and 735 imaging modalities. Indicator dilution methods include thermodilution, dye dilution, and lithium 736 dilution. Fick methods include direct oxygen and indirect carbon dioxide based methodologies. 737

Imaging modalities include ultrasound, MRI and nuclear scintigraphy. 738

739

Direct Fick method 740

Adolf Fick first postulated that CO can be obtained by assuming that the conservation of 741

• mass is valid in the body. Hence, CO is the oxygen consumption ( VO2 ) divided by the oxygen 742 content difference between artery (CaO2) and vein (CvO2): 743

• VO (FiO × V )− (FeO × V ) Cardiac output = 2 = 2 i 2 e , 744 CaO2 − CvO2 CaO2 − CvO2 where FiO2, FeO2, Vi, Ve denote inspiration and expiration oxygen fraction and inspiration and 745 expiration volume. The technique has been more widely used after the development of a 746 pulmonary catheter and advances in oxygen concentration measurement, and deemed the 747 reference standard for CO measurements. 748

In order to minimize the risks of instrumentation for the direct Fick method, indirect 749 methods have been developed, which can use carbon dioxide instead of oxygen, providing a 750 non-invasive, quick, and easier CO measurement. Similar to the direct Fick method, CO is also 751 measured by assuming the conservation of mass: 752

• VCO Cardiac output = 2 753 CaCO2 − CvCO2

Moreover, cardiac output measurement can be simplified by allowing the subject to rebreathe: 754

• • VCO VCO Cardiac output = 2nonrebreathe = 2rebreathe 755 CaCO2nonbreathe − CvCO2nonbreathe CaCO2rebreathe − CvCO2rebreathe

756

Because the venous side possesses larger carbon dioxide stores in the body and a 757 relatively slower response time, the difference between two CvCO2 is negligible. Hence one can 758 acquire CO after rearranging the equation: 759

• Δ VCO Cardiac output = 2 760 ΔCaCO2

To further simplify the measurement, CaCO2 can be estimated by: 761

, CaCO2 = (6.957[Hb] + 94.864)× log(1+ 0.1933PACO2 ) 762 where PACO2 denotes alveolar carbon dioxide partial pressure which can be extrapolated from 763 end-tidal carbon dioxide partial pressure. 764

765

Indicator method 766

The original method by Stewart involves adding a known concentration (C1) and volume 767

(V1) of sodium chloride to a central vein and collecting the blood in the femoral artery to 768 measure the concentration in the blood (C2). Also, an electrical resistance sensor is placed in the 769 contralateral femoral artery to detect the arrival of the injectate. Once C2 is measured, one can 770 acquire the blood volume over the duration (V2): 771

Solute = C1V1 = C2V2 . 772

Then CO can be calculated by dividing the blood volume by the duration(Stewart 1897): 773

V C ⋅V Cardiac output= 2 = 1 1 774 t C2 ⋅t

775

The main weakness of this method is omitting the concentration change over time as an 776 explicit curve instead of a stepwise method because the blood flow is laminar and the dilution 777 commenced from injection through sampling (Hamilton et al. 1932). Hence, Hamilton revised 778 the theory in 1928 by using the area under curve to better describe the concentration changes 779 over time (Hamilton et al. 1928). 780

V C V Cardiac output= 2 = 1 1 781 t ∫ c(t)dt t

Following this, CO has been measured with indocyanine green as indicator for decades 782

(Miller et al. 1962). 783

In 1954, Fegler and colleagues developed an intermittent bolus pulmonary artery 784 thermodilution method based on thermodynamics (Fegler 1954). In brief, Fegler substitutes 785 indocyanine green with cold solutions assuming the thermal energy is preserved in the 786 circulation. Hence, the “negative” heat of the injectate corresponds to blood before injection 787 would be the same as the blood volume over the duration compare to blood after equilibrium: 788

V1σ1ρ1(TB -T1)= V2σ BρB (TB -T2 ), 789 where σ, ρ denote specific heat and specific gravity and V1 and T1 for volume and temperature of 790 the injectate, TB for temperature of the blood, T2 for blood temperature after equilibrium, V2 for 791 blood volume over time. Then again CO can be acquired by dividing the blood volume by 792 duration: 793

V σ ⋅ ρ ⋅(T -T ) V Cardiac output = 1 ⋅ 1 1 B 1 = 2 794 t σ B ⋅ ρB ⋅(TB -T2 ) t

To take the same consideration when using dye, area under the curve is used: 795

V ⋅(T -T )⋅ K V Cardiac output = 1 B 1 1 = 2 , 796 ∫ ΔTB dt t t

σ1ρ1 where K1 denote . 797 σ BρB

There are several inherent errors for the thermodilution technique such as gain of 798 temperature before, during and after injection by error in injectate volume, catheter dead space or 799 unintentional warming by room temperature or tissue, all of which would overestimate the CO 800

(Reuter et al. 2010). Other than mentioned above, patient status could also affect the accuracy 801 and precision of this technique. For example, left-to-right shunt and differential decrease of 802 detector site blood flow during one lung ventilation would underestimate the CO (Reuter et al. 803

2010). On the other hand, tricuspid regurgitation would both over- and underestimate the CO 804

(Reuter et al. 2010). Finally, CO and baseline blood temperature fluctuation could also affect the 805 measurement (Reuter et al. 2010). 806

In order to reduce the risks related to pulmonary artery catheterization, a 807 trans-cardiopulmonary method has been investigated. In fact, in the very beginning of CO 808 measurement development, the trans-cardiopulmonary approach was used by Stewart (Stewart 809

1897). Basically, trans-cardiopulmonary methods use the same concept as the pulmonary artery 810 method but inject the indicator into the central vein and collect the data in the central artery 811 instead. Hence, the same inherent errors apply. Numerous studies have shown good correlation 812 and agreement between the two methods despite the physiological differences present in the 813 methods (Reuter et al. 2010). For example, the trans-cardiopulmonary method uses the left 814 ventricle instead of right for measurement, has more injectate loss, as well as recirculation of 815 injectate, but less effects from respiratory oscillations. 816

Besides thermal dilution methods, lithium has been investigated as an indicator partly to 817 reduce the influence of heat loss. In fact, numerous studies have proven the accuracy between the 818 methods and that peripheral veins and arteries can be used (Reuter et al. 2010). It should be 819 noted that lithium only distributes in the plasma, thus hemoglobin concentrations must be entered 820 to estimate packed cell volume as well as sodium concentration for baseline voltage of the 821 lithium dilution sensor interface. 822

Pulse contour method 823

To derive CO from the pulse contour method, mathematical modeling of how blood 824 travel in the vessel after leaving the ventricles has been studied. Among different models, the 825

Windkessel model has been used extensively. There are two basic assumptions in the model; first, 826 conservation of mass: the net gain of the blood volume equals to the net loss of the blood volume 827 in the vessel during the cardiac cycle; and second, the effect of compliance is predictable (Thiele 828 et al. 2015). 829

The initial work estimates stroke volume from the arterial waveform by using two 830 elements of the Windkessel model (Warner et al. 1953). This model includes compliance on top 831 of resistance to describe the nature of the pulsatile blood flow in the aorta and arteries compared 832 to the steady laminar flow in the model using the Hagen-Poiseuille Law of Friction. As the 833 vessel expands and keeps part of the blood during systole, it also contracts and expels the stored 834 blood during diastole (Sagawa et al. 1990). Stroke volume is divided into QS (systolic outflow) 835 and QD (diastolic outflow). QD is proportionate to end-systolic pressure (Pmd) (Warner et al. 836

1953): 837

QD = k × Pmd 838

Where k denotes a constant, which includes resistance and compliance. Next, assuming the 839 resistance is constant through the cardiac cycle, the ratio between Qs and QD equals to the ratio 840 between the area under the pressure-time curve of each (As and AD). After integrating the two 841 equations, SV can be derived from the pulse contour as: 842

SV = k × Pmd (1+ AS / AD ) 843

Once the k is known by calibrating with another measurement method, a continuous 844 measurement is provided. 845

Later on, the model evolved to include the wave reflection, the impedance which is the 846 oscillating resistance to a pulsatile flow, and inertance which is the pressure required for flow 847 rate changes (Westerhof et al. 2009). 848

The Modelflow technique uses three elements of the Windkessel model which includes 849 impedance to derive stroke volume (Wesseling et al. 1993). 850

The pulse index continuous cardiac output (PiCCO) system allows both transthoracic 851 thermodilution measurement (PiCCOTD) and continuous pulse contour analysis (PiCCOc). For 852 later versions of PiCCO, aortic impedance and instantaneous pressure changes are taken into the 853 calculation of SV and only the systolic portion of the pulse contour are used. Because of the 854 differences among individual impedance values, calibration to the transthoracic measurement is 855 recommended (Godje et al. 2002). 856

The PulseCO system also uses three elements of the Windkessel model. First, the 857 machine uses a diagram to estimate central arterial pressure from the peripheral arterial pressure. 858

Then, by corresponding pressure changes to the arterial compliance curve, a volume-time 859 waveform is generated. Stroke volume is derived from the volume-time waveform and therefore 860

CO after being adjusted for heart beat duration. Calibration with LiDCO measurement is 861

38 recommended by the company because the arterial compliance curves differ in the scale despite a 862 similar shape (Linton & Linton 2001). 863

Instead of bringing data into a model based on theory, FloTrac/Vigileo system uses an 864 empirical mathematic model to derive SV regardless of whether or not the model fits a 865 physiological theory (Pratt et al. 2007). In this system, only two variables are used: standard 866 deviation of mean arterial pressure over 20 seconds (σ AP ) and a conversion factor (k) which 867 includes HR, body surface area, compliance, skewness and kurtosis, MAP, and standard 868 deviation over 60 seconds. Because the model is based on empirical data, no calibration is 869 required. 870

Ultrasound-based method 871

Echocardiography uses ultrasound to measure the doppler shift of blood to extrapolate 872 blood flow velocity in the ascending aorta or left ventricle outflow tract though the equation: 873

f × c Vb = 874 2 × f0 × cosθ with blood velocity (Vb), frequency shift (f), sound wave velocity in the blood (c), ultrasound 875 beam frequency (f0 ) and the angle between the ultrasound beam and red blood flow (cosθ). Then 876

SV can be calculated by multiplying blood flow time velocity integral by the cross-sectional area 877 of the vessel being interrogated. When using descending aorta, the distribution of cardiac output 878 proximally needs to be corrected by a calibration factor. 879

There are two ways of acquiring cross-sectional area of the descending aorta: large 880 population databases and M-mode echocardiography measurements. When using databases, 881 individual variation could contribute to error. On the other hand, M-mode echocardiography is 882 operator dependent since the angle of insonation and movement of the probe could lead to 883 different measurements. Moreover, the cross-section area of the aorta changes over the cardiac 884

39 cycle so the timing of measurements could also affect the measurement. Nevertheless, ultrasound 885 based techniques have shown minimal bias and good agreement with thermodilution techniques 886

(Dark & Singer 2004; Thiele et al. 2015). 887

888

Thoracic electrical impedance method 889

This technique uses the electrode to detect the electrical impedance changes in the thorax 890 and assumes that the only factor contributing to these changes is the changing blood volume 891 from the beating heart. Hence SV can be derived from such signals. However, many 892 confounding factors can attribute to the signal such as motion, abnormal anatomy or pathology 893 and arrhythmias. Therefore this technique is more suitable for trending changes and is not 894 necessarily of use for diagnosis (Raaijmakers et al. 1999). 895

896

Validation in dogs 897

Direct fick method 898

The Fick method has been deemed as the gold standard of CO measurement in the 899 literature to evaluate other methods. Hence only a few studies investigate the accuracy of the 900 direct Fick method when compare to a direct flow measurement. Nevertheless two studies done 901 in 1950 compared directly measured blood flow by rotameter to the results acquired from direct 902

Fick method and showed minimal bias with good correlation of r= 0.96 (Huggins et al. 1950; 903

Seely et al. 1950). 904

905

Thermodilution method 906

Due to the complexity of acquiring CO through the direct Fick method, thermodilution 907 has been used to replace the direct Fick method as clinical standard. Actually dogs were used in 908 the study when Fegler first published the method in 1954 (Fegler 1954). In the study, 909 thermodilution was compared to the direct Fick method and showed minimal bias. Moreover, in 910 another study comparing thermodilution to Fick where heated saline was injected into blood 911 instead of cold saline minimal bias was demonstrated (Khalil et al. 1966). 912

913

Lithium dilution method 914

The first published application of the lithium dilution measurement of CO (LiDCO) in 915 dogs is in 2001 (Mason et al. 2001). In this study, they compared lithium dilution measurements 916 to thermodilution measurements (TD) in halothane anesthetized dogs under four different 917 hemodynamic conditions: light plane of anesthesia; dobutamine infusion; moderate - deep plane 918 of anesthesia; deep plane of anesthesia or occlusion of the caudal vena cava. They also tested 919 two different doses of lithium. They concluded that with either high or low dose lithium, 920 clinically relevant ranges of CO (<5 L minute-1) or pooled data all showed excellent correlations 921 to TD, being more than 0.97 (Mason et al. 2001). The bias of pooled data and CO, less than 5 L 922 minute-1, between LiDCO and TD is 0.084 ± 0.465 and 0.002 ± 0.245 L minute-1. Later on, the 923 same research group validated that use of a peripheral vein for injection of lithium correlates 924 well with central vein injection. In addition, background serum lithium concentration has limited 925 clinical significance on the measurement (Mason et al. 2002a; Mason et al. 2002b). 926

Another study compared LiDCO to TD in sevoflurane anesthetized dogs under three 927 hemodynamic conditions: normodynamic (one minimum alveolar concentration [MAC]); 928

41 hypodynamic (two MAC); hyperdynamic (noradrenaline infusion). The study showed a bias of 929

-0.57 ± 0.96 L minute-1 m-2 during hypodynamic conditions; -0.05 ± 0.89 L minute-1 m-2 during 930 normodynamic conditions; -0.03 ± 2.42 L minute-1 m-2 during hyperdynamic conditions; and 931

-0.11 ± 1.55 L minute-1 m-2 when all data was pooled together (Morgaz et al. 2014). In contrast to 932 the previous study by Mason, this study showed a higher bias, especially in hypodynamic 933 conditions, although they had a higher standard deviation overall. 934

935

Pulse contour methods 936

PulseCO 937

The first research investigating the PulseCOTM (PulseCO) in dogs was performed in 2005. 938

In this study, PulseCO was compared to LiDCO in isoflurane anesthetized dogs after calibration 939 with LiDCO during six different hemodynamic conditions: light plane (1-1.5 MAC); deep plane 940

(2-2.5 MAC); dopamine or dobutamine infusions of 7µg kg-1 min-1; dopamine or dobutamine 941 infusions to achieve MAP between 65-80 mmHg. The overall correlation coefficient was 942 moderate (r=0.6289). The limits of agreement and bias, in the same order as mentioned above, 943 were 0.55 to 1.82; 1.24 to 4.12; 0.24 to 0.81; 0.46 to 1.51; 0.3 to 0.98; 0.36 to 1.18 L minute-1 944

(Chen et al. 2005). In the study methods, PulseCO generally tracked the changes in CO along 945 with LiDCO despite the overestimation with PulseCO during deep planes of anesthesia. 946

Nevertheless, the authors of this study recommended recalibration whenever significant 947 hemodynamic or vascular changes occurred. 948

Similar overestimation during hypodynamic conditions was also shown in mild to 949 moderate (Dyson & Sinclair 2006) and severe hemorrhage(Cooper & Muir 2007) canine 950 experimental models. Despite the findings that the PulseCO did not require recalibration in four 951

42 hours of use in a human critical care unit, (Cecconi et al. 2008) a small-scale veterinary clinical 952 case series showed a large percentage of error despite an acceptable bias in natural occurring 953 systemic inflammatory response syndrome in dogs (Duffy et al. 2009). Hence, recalibration is 954 recommended with the PulseCO methods for enhanced accuracy. 955

956

PiCCO 957

The first research investigating the application of both PiCCOTD and PiCCOc in dogs was 958 performed in isoflurane anesthetized research dogs (Shih et al. 2011). The research compared 959 both PiCCO systems to LiDCO with three different hemodynamic conditions: intermediate 960

(0.9-1.4% ETIso); high (0.9-1.4% ETIso with artificial pacing heart rate of 120 bpm); and low 961

(2.1-2.7% ETIso). This study compared the manufacturer recommended femoral artery use with 962 measurement in the metatarsal artery. Nevertheless, PiCCOc at both sampling sites resulted in 963 low precision despite an acceptable accuracy with femoral artery site (Shih et al. 2011). 964

In another study, PiCCOTD not only correlated well with traditional thermodilution 965 measurements (r=0.915) but also showed minimal bias (-0.04 ± 1.19 L minute-1) despite 966 moderate precision. Moreover PiCCOc tracked along well with PulseCO (Morgaz et al. 2014). 967

968

FloTrac/Vigileo 969

Only two published research studies the application of FloTrac/Vigileo in anesthetized 970 dog (Valverde et al. 2011; Bektas et al. 2012). These studies inputted 15 or 20 years old as age 971 and converted height from body surface area, while measuring CO under different hemodynamic 972 conditions. Nevertheless both of the studies showed poor accuracy and precision and deemed the 973 modality unreliable in monitoring CO in dogs. 974

Indirect Fick Method 975

NICO (Non-invasive cardiac output) 976

The only commercial machine uses indirect Fick method to measure CO is the NICO® 977 by Novametrix Medical Systems Inc. NICO® has been compared to the thermodilution method 978 and LiDCOTM in dogs. NICO® showed precision of 13.8%, bias of -1.4% and r= 0.93 in the study 979 comparing it to the thermodilution method (Haryadi et al. 2000). When compared to LiDCOTM, 980

NICO® showed r=0.888, relative error of 2.4± 24.7% and mean difference of measurement from 981

LiDCOTM to NICO® is 1.11 time NICO® (Gunkel et al. 2004). However, NICO® produced 982 significant lower values when compared to thermodilution in another study and showed 983 percentage error of 56 % (Yamashita et al. 2007). The authors attributed the difference to the size 984 of the dogs. 985

Thoracic electrical impedance method 986

Thoracic electrical impedance changes on SV prediction has been used in dogs (Ito et al. 987

1976). Perfusion of the aorta with sinusoidal and pulsatile wave blood flow in two dogs induced 988 changes in the recorded impedance. With this method, the relative instead of actual SV is 989 acquired and the frequency of the sinusoidal wave greatly affects the results. Several in vivo 990 studies also show good correlation between electrical impedance to indicator dilution 991 measurement under different hemodynamic conditions despite a certain degree of bias (Hill et al. 992

1976; Kiesler et al. 1990; Sherwood et al. 1991; Adamicza et al. 1994). Nevertheless, in a more 993 recent study comparing electrical impedance to thermodilution measurements under different 994 cardiac outputs by manipulating sevoflurane concentration and dobutamine infusion, high limits 995 of agreement of -0.58 ± 1.56 L min-1 and percentage error of 75.4% were demonstrated 996

(Yamashita et al. 2007). It is important to point out that the different module, electrodes 997 placements, and recording might account for part of the difference. 998

999

Ultrasound-based method 1000

Transthoracic Doppler CO measurement of pulmonary blood flow (DP) has been 1001 compared to TD in dogs and correlates better than aortic blood flow (DA). The bias of TD to DP 1002 and DA is -0.04 ± 0.22 (95% CI=-0.11 to 0.03) L minute-1 and -0.87 ± 0.54 (95% CI= -0.69 to 1003

-1.04) L minute-1. The limit of agreement of TD to DP and DA is -0.49 (95% CI= -0.36 to -0.61) 1004 to 0.4 (95% CI= 0.52 to 0.28) and -1.96 (95% CI= -1.65 to -2.26) to 0.22 (95 CI= 0.52 to -0.08) 1005

L minute-1 (Lopes et al. 2010). 1006

Besides traditional echocardiography, USCOMTM is a commercial modality being 1007 developed and validated in dogs. Critchley and colleagues compared USCOMTM to a surgically 1008 instrumented ultrasound flow probe around the aorta in halothane anesthetized dogs with various 1009 levels of CO created by variations in inhalant concentrations and dopamine infusions. Instead of 1010 using the human database they measured the aortic diameter during the surgery. In the study, 1011 they showed bias of -0.01 (95% CI= -0.34 to 0.31) L minute-1 and percentage of error of 13% 1012

(Critchley et al. 2005). 1013

Another study comparing the USCOMTM to TD in isoflurane anesthetized dogs measured 1014 the aortic diameter before anesthesia through traditional echocardiography. Furthermore, they 1015 measured CO under four different hemodynamic conditions: baseline (0.5-1.5% isoflurane); deep 1016 anesthesia (2-3.5% isoflurane); a colloid solution infusion (right atrial pressure more than 15 1017 mmHg); dobutamine infusion (5µg kg-1 min-1). The overall bias when measured from the 1018 subxiphoid and thoracic inlet windows is -0.03 ± 0.73 and -0.2 ± 0.8 L minute-1 respectively. The 1019

45 limit of agreement when measured from subxiphoid and thoracic inlet is -1.47 to 1.41 and -1.76 1020 to 1.36 L minute-1 with a percentage of error of ± 46% and ± 53% (Scansen et al. 2009). The 1021 variations in measurement seem smaller during low CO conditions, which is more commonly 1022 encountered in those patients that may benefit from CO monitoring. 1023

Transesophageal echocardiography (TEE), in which the ultrasound probe is passed orally 1024 into the thoracic esophagus to image the heart base more accurately, has gained growing interest 1025 in human medicine. In dogs, however, accuracy seems dependant on the hemodynamic 1026 conditions. In a study comparing TEE to thermodilution measurements under different 1027 sevoflurane concentrations in dogs, a limit of agreement of 0.03 ± 0.26 L minute-1 and 1028 percentage of error of ± 12.3% was noted (Yamashita et al. 2007). Another study compared TEE 1029 to a surgically placed ultrasound probe around the aorta and TD under moderate to severe 1030 hemorrhage and resuscitation. The study shows TEE overestimates CO in general and has a 1031 moderate correlation despite a good correlation during severe hemorrhage (de Figueiredo et al. 1032

2004). 1033

1034

Summary of cardiac output methods 1035

The direct Fick method is still deemed as the gold standard. However, for clinical or 1036 even practical research use, LiDCO is well validated and confirmed as being reliable in dogs. 1037

For pulse contour analysis methods, the low precision would generally limit the application and 1038 some techniques (FloTrac/Vigileo) have no applicability in dogs due to algorithms based on 1039 humans used by this technology. With proper calibration, especially after significant 1040 hymodynamic changes, PulseCO matched well with LiDCO except for during hypodynamic 1041 conditions. For the indirect Fick method, NiCO is an acceptable alternative except in smaller 1042

46 size dogs. Because of different modules and models being used, electrical impedance has not 1043 shown great potential for CO measurement in dogs. Hence, further studies are warranted. 1044

Ultrasound based methods have shown great potential but measurements largely rely on 1045 personnel skill and experience. 1046

1047

LITERATURE REVIEW SUMMARY 1048

This study will provide insight regarding the usefulness of co-administration of 1049 midazolam with propofol or alfaxalone versus other studies that administer midazolam after the 1050 initial propofol bolus with various pre-medication. The results will contribute to the debate of 1051 the cardiovascular benefits of co-administration of midazolam with either induction agent, by 1052 measuring cardiac output and direct arterial blood pressure. This study will also provide a 1053 direct comparison between propofol and alfaxalone for TIVA in pre-medicated mechanically 1054 ventilated dogs during a diagnostic procedure, without significant surgical insult. Suggestions on 1055 recovery quality and differences can be made without the impact of pain or discomfort on the 1056 recovery results. It is anticipated that these results will be applicable to everyday clinical 1057 canine anesthesia. 1058

1059

1060 1061 1062 1063 1064 1065 1066 1067

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1563

1564

Table 1. Summary of cardiovascular effects of induction with alfaxalone in dogs. 1565 Doses Baseline 1 minutes 5 minutes [mean(SD)] References [mean(SD)] [mean(SD)] [mean(SD)] (mg kg-1) (Rodriguez 4.15(0.7) 127(16) 178(13)* 160(14)* et al. 2012) (Maney et al. 2.6(0.4) 121(25) 144(46) 145(33) 2013) (Amengual 1.5 104(22) NA 114(28) HR (bpm) et al. 2013) 2 120(21) 155(18) 143(17) (Muir et al. 6 112(28) 158(33)* 150(20)* 2008) 20 128(33) 162(16)* 131(19) (Ambros et 2 87(21) NA 100(21) al. 2008) 4.15(0.7) (Rodriguez 126(8) 113(13) 103(6)* et al. 2012) (Maney et al. 2.6(0.4) 102(11) 106(10) 81(11) 2013) (Amengual MAP 1.5 98(15) NA 72(13)* et al. 2013) (mmHg) 2 120(12) 115(18) 114(9) (Muir et al. 6 113(12) 122(28) 101(18)* 2008) 20 123(13) 94(29)* 65(13)* (Ambros et 2 76(7) NA 67(6) al. 2008) CI 4.15(0.7) (Rodriguez 3.33(0.7) a 4.1(0.7)*a 3.84(0.6)*a (L minute-1 et al. 2012)

m-2) = a or (mL kg-1 -1 2 229(60) b 245(53) b 231(77) b minute ) = b (Muir et al. 2008)

6 218(62) b 238(80)* b 222(71) b 20 246(71) b 221(60)* b 190(64)* b (Ambros et 2 155(45) b NA 164(47) b al. 2008) SVR/ 4.15(0.7) (Rodriguez 7526(1700 a 5181(923) a 4984(632) a SVRI et al. 2012) (dynes 2 2865(1207) b 2537(980) b 2906(1739) b (Muir et al. second cm-5 6 2830(905) b 2803(1057) b 2441(1004) b -2 2008) m ) = a 20 2657(654) b 2180(664) b 1781(350) b or (dynes (Ambros et second cm-5) 2 1826(410) b NA 1580(591) b al. 2008) = b

* Indicates paramters that are significantly different from baseline. 1566 1567 1568 1569 1570 1571 1572 1573 1574 1575 1576

Table 2. Induction Doses and Quality with Alfaxalone. 1577

Dose (SD) Induction Subjects Premedication Reference (mg kg-1) quality Research (Ferre et al. 2* None N/A beagles 2006) Client-owned Acepromazine/M (Suarez et al. 1.9(0.07) Smooth healthy dogs orphine 2012) Buprenorphine/A Client-owned cepromazine or Good to (Herbert et al. 1.5(0.57) healthy dogs Dexmedetomidin smooth 2013) e Crossbred Acepromazine/H (Ambros et al. 2* Smooth research dogs ydromorphone 2008) 1.2(0.4) Client-owned Medetomidine 1.2(0.4) healthy dogs Butorphanol 5.8% poor, 17.6% fair, (Maddern et al. 70.5% Medetomidine/bu 2010) 0.8(0.3) excellent, torphanol 5.8% unknown

Client-owned 1.91 clinical (Psatha et al. Methadone N/A (0.29) compromised 2011) dogs Research (Rodriguez et al. 4.15(0.7) None Smooth beagles 2012) Smooth (one Client-owned Acepromazine/m needs (Amengual et al. 1.5* healthy dogs eperidine additional 2013) dose)

Crossbred Good to (Maney et al. 2.6(0.4) None research dogs smooth 2013) Crossbred Good to (Muir et al. 2* None research dogs smooth 2008) Client-owned Acepromazine/ healthy less than (O'Hagan et al. 1.7(0.3) atropine/ Smooth 12 weeks of age 2012) morphine dogs 1578 * Indicates fixed bolus doses instead of titration to effect. 1579 1580

CHAPTER II

INDUCTION DOSE AND RECOVERY QUALITY OF PROPOFOL AND

ALFAXALONE WITH OR WITHOUT MIDAZOLAM CO-INDUCTION

FOLLOWED BY TOTAL INTRAVENOUS ANESTHESIA IN DOGS

SUMMARY

Objective To compare the induction dose and recovery quality of propofol and alfaxalone with or without midazolam followed by total intravenous anesthesia (TIVA) in dogs.

Study design Prospective, randomized, incomplete Latin-square crossover, blinded trial.

Animals Ten research dogs weighing a mean (SD) of 24.5 (3.1) kg.

Methods Dogs were randomly assigned to four treatments, Propofol (P) with saline (S),

P-S; Alfaxalone (A) with S, A-S; P with Midazolam (M) P-M; A-M. Fentanyl (7 µg kg-1,

IV) was administered 10 minutes prior to an IV bolus of P (1 mg kg-1) or A (0.5 mg kg-1) followed by M (0.3 mg kg-1, IV) or S and additional boluses (Add-Dose) of P or A every

6 seconds for intubation, followed by maintenance with P/A TIVA. Quality of sedation

(SedQ), induction (IndQ), maintenance (AnesQ), extubation (ExtQ) and recovery (RecQ) were scored. Total dose (TotalD) and Add-Dose of P or A, TIVA rates, time from sedation to TIVA discontinuation (T1), times from TIVA discontinuation to extubation

(T4) and standing (T5) were recorded. Analysis included a general linear mixed model with post hoc analysis (p < 0.05).

Results The IndQ was better in A-M versus A-S; P-M versus P-S, and A-M versus P-S.

The TotalD was lower in P-M versus P-S and A-M versus A-S. The Add-Dose were

74 fewer in A-M versus A-S; P-M versus P-S; and A-M versus P-S. The TIVA rate of P-M was lower than P-S but similar between A-M and A-S. Scores for SedQ, ExtQ, and RecQ, and times for T1 and T4 were similar between treatments. The T5 was longer for A than

P, but similar within A or P treatments.

Conclusions and clinical relevance In fentanyl sedated dogs, M improved IndQ and reduced TotalD and Add-Dose for both P and A, but only reduced TIVA rate of P.

Keywords alfaxalone, co-induction, dog, midazolam, propofol

INTRODUCTION

The optimal anesthetic induction agent in small animal practice would be cost effective, easy to administer intravenously (IV) without pain or irritation to allow for a smooth induction with endotracheal intubation, without any negative cardiopulmonary side effects, and with a fast and calm recovery. Propofol (P) and alfaxalone (A) are two commonly used induction agents in small animals, which have good qualities; however, anesthetic induction may not always be smooth, necessitating higher induction doses and increasing the negative cardiopulmonary side effects. Co-induction agents can be used with propofol or alfaxalone with the goal of promoting a smooth induction, reducing the induction dose, and thereby the negative cardiopulmonary effects.

The main co-induction agents used in veterinary medicine are diazepam, midazolam, lidocaine, and ketamine. The veterinary literature indicates both positive and negative findings of induction dose reduction with co-induction agents (Lerche et al.

2000; Ko et al. 2006; Braun et al. 2007; Jolliffe et al. 2007; Covey-Crump & Murison

2008; Mair et al. 2009; Robinson & Borer-Weir 2013; Sanchez et al. 2013;

Martinez-Taboada & Leece 2014). The variability in results with benzodiazepines in dogs is associated with differences in premedication agents, which benzodiazepine and dose is used, as well as order and speed of administration. With midazolam co-induction with propofol, the dose reduction is most consistent at doses of 0.2-0.5 mg/kg and when the midazolam is given after an initial bolus of propofol (Robinson &

Borer-Weir 2013; Sanchez et al. 2013). An observational study investigated the dose reduction effects of using of midazolam as a co-induction agent with alfaxalone in dogs

(Seo et al. 2015). Both alfaxalone and propofol have the advantage of pharmacologic

76 profiles that allow administration for total intravenous anesthesia (TIVA). In general, recovery from propofol TIVA has been described as smooth and excellent (Keegan &

Greene 1993; Ambros et al. 2008; Suarez et al. 2012). However, the recovery quality of alfaxalone is controversial. Most studies state that the overall recovery quality with alfaxalone is good to excellent (Ambros et al. 2008; Muir et al. 2008; Psatha et al. 2011;

Suarez et al. 2012; Herbert et al. 2013). Recovery from alfaxalone TIVA has been reported as comparable to propofol TIVA (Ambros et al. 2008; Suarez et al. 2012).

Recovery from alfaxalone induction was better than etomidate (Rodriguez et al. 2012) or diazepam/fentanyl induction (Psatha et al. 2011) when followed by isoflurane maintenance. However, dogs receiving alfaxalone induction followed by sevoflurane for MRI had poorer recovery scores compared to those induced with propofol (Jimenez et al. 2012). With alfaxalone, dogs may be sensitive to external stimulation (Ferre et al.

2006) or demonstrate tremors, rigidity, and myoclonus at recovery (Maney et al. 2013).

Hence pre-medication as well as a quiet and undisturbed recovery are recommended by the manufacturer (Jurox Pty Ltd, Australia). To the authors knowledge, a direct comparison of the TIVA maintenance quality and recovery characteristics of propofol and alfaxalone TIVA when co-induction with midazolam in fentanyl pre-medicated dogs for diagnostic computerized tomography (CT) or magnetic resonance imaging (MRI) is not available.

The hypothesis of this study is that there are no differences regarding the induction dose, quality, TIVA dose, ease of maintenance, and overall recovery characteristics between propofol and alfaxalone with or without midazolam co-induction in fentanyl sedated dogs.

MATERIALS AND METHODS

Animals

All procedures were approved by the Animal Care Committee, University of

Guelph, and followed the Canadian Council on Animal Care Guidelines. Ten healthy research crossbred hound dogs, mean (range) age 3.4 (1.9 – 5.5) years, mean (SD) weight

24.5 (3.1) kg were used. Health status was based on general physical examination, complete blood count, and biochemistry panel.

Study Design

The sample size was calculated to detect 30% difference of propofol or alfaxalone induction dose with type 1 error of 0.05 and power of 80%. A minimum of four dogs in each treatment was needed. This study was a prospective, blinded, randomized, incomplete Latin-square crossover research trial with at least a seven days washout between five separate anesthetic events that included three MRI and two CT scans. A random sequence was generated using a computer algorithm (GraphPad Prism, CA, USA) to ensure blinded allocation of dogs between the treatments. Papers containing the anesthetic treatment allocation of dog for the research day were organized and opened by anesthesia research technicians. The research technicians also prepared the syringes for the randomized drugs for induction, additional doses, constant rate infusions, as well as adjustment of TIVA doses. Drapes were used to cover the syringes and infusion lines to prevent either anesthetist (MS, PL) from identifying the drug used.

Dogs underwent MRI (first, third and fourth anesthetic event), a CT-guided intervertebral disc injection of gelified ethanol (CT1; second event) in a parallel but

78 unrelated study (Mackenzie et al. 2016) and a CT scan without injection (CT2; fifth event) while maintained on propofol or alfaxalone TIVA. The results of the cardiopulmonary variables are presented elsewhere.

Anesthesia and instrumentation (see Figure 1)

The dogs were fasted for at least 12 hours but given free access to water prior to general anesthesia. Topical local anesthetic cream (Maxilene, RGR Pharma Ltd.,

Canada) was applied pre-emptively to the clipped area over the planned catheterization sites of the cephalic vein and dorsal pedal artery at least ten minutes before. A 20-gauge catheter (Insyte-W; Becton Dickinson Infusion Therapy Systems, UT, USA) was inserted into a cephalic vein and 0.1 mg kg-1 of meloxicam (Metacam® 5 mg mL-1, Boehringer

Ingelheim Canada LTD, Canada), was administered intravenously prior to sedation.

With the dogs in lateral recumbency and monitored for cardiopulmonary parameters as part of a second study, fentanyl was administered at 7 µg kg-1, IV (Fentanyl

50 µg mL-1, Sandoz Canada Inc., Canada). Sedation was scored on each dog 10 minutes later, prior to administration of the induction agent (SedQ, Sedation Quality Score

Appendix 1). Dogs were randomly assigned to one of four treatments for anesthetic induction with propofol (Propofol 10 mg mL-1, Pharmascience Inc, Canada) or alfaxalone

(Alfaxan® 10 mg mL-1, Jurox Pty Limited, Australia) with or without midazolam

(Midazolam 5 mg mL-1, Pharmaceutical Partners of Canada Inc, Canada) or normal saline

(0.9% Sodium Chloride, Hospira, Canada), and maintained by TIVA with the respective injectable agent as follows: Treatment P-M: propofol (1 mg kg-1, IV) and midazolam (0.3 mg kg-1, IV); Treatment A-M: alfaxalone (0.5 mg kg-1, IV) and midazolam (0.3 mg kg-1,

IV); Treatment P-S: propofol (1 mg kg-1, IV) and equal volume of saline (0.06 mL kg-1,

IV); Treatment A-S: alfaxalone (0.5 mg kg-1, IV) and equal volume of saline (0.06 mL kg-1, IV). Initial doses of propofol or alfaxalone were administered as a bolus, flushed with 2 mL of saline, IV, and then the midazolam or equal volume of saline was immediately administered. An additional 25% of the bolus dose of propofol or alfaxalone was administered as a bolus every 6 seconds upon request until successful endotracheal intubation could be performed (MS). No attempts to intubate were made until confirmed loss of lateral palpebral reflex and then relaxation of jaw tone.

Induction and intubation scores based on quantity of additional boluses required for intubation (Add-Dose), speed of relaxation, response to laryngoscope placement, and endotracheal intubation, or presence of excitement was assessed and scored by the same researcher unaware of treatments allocation (MS) (IndQ, Induction and Intubation

Quality Score Appendix 2).

Once intubated, TIVA with the respective injectable was administered by syringe pump (Medfusion 3500, Smiths Medical Canada, Canada; Graseby 3500 Anesthesia

Pump; Smiths Medical International Ltd, UK) for maintenance at the following initial rates: 250 µg kg-1 minute-1 of propofol or 70 µg kg-1 minute-1 of alfaxalone. Dogs were connected to a small animal anesthesia machine with rebreathing circuit (Universal

F-Circuit; Dispomed, Canada) using an oxygen flow of 1.5-2.5 L minute-1 with commencement of entidal carbon dioxide partial pressure (PE´CO2) and respiratory rate

(fR) measurements. Respiratory rate was acquired from capnography and rectal temperatures (Temp; ˚C), which were measured and recorded at each blood gas determination. Dogs were allowed to breathe spontaneously for 15 minutes in lateral

80 recumbency. A manual breath was administered if the dog did not breathe spontaneously for more than 30 seconds. After 15 minutes of spontaneous ventilation,

-1 mechanical ventilation was initiated with a tidal volume of 10 mL kg and fR adjusted to

PE´CO2 of 35 to 45 mmHg and the dog transferred to initiate the CT or MRI. Depth of anesthesia was evaluated and kept at the lightest possible plane by a researcher unaware of treatment allocation (PL). For minor changes in depth, TIVA rate adjustments by 25

µg kg-1 minute-1 for propofol and 7 µg kg-1 minute-1 for alfaxalone were made. When a rapid increase in depth was warranted, boluses of propofol (0.25 mg kg -1) or alfaxalone

(0.125 mg kg -1) were administered. Anesthetic depth was assessed with physical signs every 5 minutes; indications of inadequate depth included movement, brisk palpebral reflex, increased jaw tone, spontaneous blinking, or continual increases in heart rate (HR), fR, or direct arterial blood pressure. Anesthetic administration was decreased in the same fashion to increase when a progressive decrease in arterial pressure was noted in the absence of respiratory fluctuations or attempts were made to ventilate spontaneously during mechanical ventilation, in addition to lack of palpebral reflexes and jaw tone.

The ease of TIVA anesthesia maintenance quality was scored every 5 minutes throughout

(AnesQ, see Anesthetic Maintenance Quality Score Appendix 3). Hypotension was defined as a mean arterial pressure (MAP) < 60 mmHg for more than 5 minutes with an appropriate anesthetic depth, and treated with an infusion of dopamine (2–10 µg kg-1 minute-1) at the researcher’s discretion if depth adjustment was not sufficient.

After the MRI or CT procedure, the dogs were transferred back to the induction area for recovery. Total anesthesia time recorded from the start of induction (T3) to

TIVA discontinuation, and from administration of sedation to TIVA discontinuation (T1)

81 were recorded. Extubation was initiated once a strong medial palpebral reflex just prior to the swallow reflex was present and recorded as time to extubation (T4) from TIVA discontinuation. Dogs were left undisturbed on the recovery table for 10 minutes and extubation quality scored immediately after extubation, and subsequently at 5 and 10 minutes before being moved to a cage (ExtQ, see Extubation Quality Score Appendix 4), where they were observed for recovery quality at 15, 30, 45 and 60 minutes after extubation (RecQ, see Recovery Quality score Appendix 5) During this period the time to standing (T5) from the end of TIVA was also recorded. The ExtQ and RecQ was scored on site by a researcher unaware of treatment allocation (PL) as well as video recorded for

10-15 seconds at each assessment. Three board certified anesthesiologists unaware of the treatments allocation, imaging modalities and time point also scored the ExtQ and RecQ through video clips (MS, AV, CM).

Statistical analysis

Statistical analyses were performed with standard computer software (SAS

OnlineDoc 9.2; SAS Institute Inc., North Carolina, USA). Normality of data was tested using Shapiro-Wilk, Kolmogorov-Smirnov, Cramer-von Mises and Anderson-Darling test (Proc UNIVARIATE, SAS). Continuous data that were not normally distributed were log transformed for analysis, unless log transformation provided no improvement to distribution. The inter-rater agreement of the ExtQ and RecQ was examined by weighted kappa statistic. A general linear mixed model was used to model the results using Proc MIXED. Fixed effect for SedQ, TotalD, Add-Dose, IndQ, and T2 was treatment, whereas for T1, T3, T4, and T5 was treatment and imaging procedure,

82 including up to two-way interactions, and for ExtQ and RecQ were treatment, imaging procedure and time, including up to three-way interactions. For AnesQ and TIVA rate, different phases (see Figure 1) including sedation, induction, procedure, and end were also considered fixed effects in addition to treatment, imaging procedure and time within phases with up to three-way interactions. No interaction was tested between phases and times within phases. Dog was considered a random effect and treated as a blocking variable. To model for the effects of repeated measures over time for TIVA rate, AnesQ, ExtQ, and RecQ on each dog, due to treatment and within a specific phase, the following correlations structures, offered by SAS, were attempted: ar(1), arh(1), sp(pow)(time), toep, banded toep, toeph, banded toeph, and un and banded un. The error structure, among those that converged, was chosen based upon the lowest Akikie

Information Criteria. Terms in the model were removed if non-significant, but preserving model hierarchy. Appropriate adjustments (Tukey or Dunnet test) for multiple comparisons were employed. Significance was set at p < 0.05. Residuals were examined to assess the ANOVA assumptions and plotted against the predicted values and the explanatory variables used in the model. Such analyses help reveal outliers, unequal variance, the need for data transformation, and other issues that need addressing.

The results are presented with adjusted mean if data are normally distributed or ordinal and adjusted median if not normally distributed based on a log transformation with 95% confidence interval (CI). The adjustment was done by applying standard

ANOVA least-squares methods to estimate and test the results as if the data were in a complete Latin-square, crossover design. When comparing between treatments, for

83 those normally distributed continuous data and ordinal data, the effect size is provided as the difference between treatments with 95% CI. The p-value is provided for those not normally distributed and log transformed.

RESULTS

All dogs recovered uneventfully from anesthesia without complications. There was a significant reduction in the TotalD with both A-M and P-M treatments with effect size (95% CI) of 0.36 mg kg-1 (0.07 – 0.65) for A-S compared to A-M and 0.52 mg kg-1

(0.22- 0.81) for P-S compared to P-M. The IndQ score was significantly better for A-M versus either A-S or P-S with effect sizes (95% CI) of 0.97 (0.25 – 1.69) and 1.69 (0.96 –

2.41) respectively. The IndQ score was also significantly better for P-M versus P-S with effect size (95% CI) of 1.41 (0.68 – 2.15). The number of Add-Dose was lower for

A-M versus A-S; P-M versus P-S; and A-M versus P-S with effect size (95% CI) of 1.9

(0.6 – 3.2); 1.8 (0.5 – 3.2); 3.0 (1.7 – 4.3). However, there were no significant differences between treatments for SedQ or T2. (see Table 3).

The overall median TIVA rates (95% CI) for P-S, P-M, A-S, and A-M were 310

µg kg-1 min (274 - 351), 268 µg kg-1 min (233 - 309), 87 µg kg-1 min (77 - 98) and 83 µg kg-1 min (74 - 94), respectively (see Table 4). The overall TIVA rate of P-M was significantly lower than P-S (p = 0.013) but there was no difference between A-M and

A-S. The TIVA rate of CT1 after induction was significantly higher than CT2 (p <

0.001). The TIVA rate of CT1 during the procedure was significantly higher than MRI

(p = 0.041) and CT2 (p < 0.001). The TIVA rate of CT1 during the procedure and the end was higher than after induction (p <0.001). The TIVA rate of MRI after the procedure was higher than after induction (p = 0.003). There was no significant difference between anesthetic events in CT2.

The AnesQ was not different between treatments (see Table 4). However, the overall AnesQ had significant changes related to the first and second CT event or MRI

85 and the different phases. After induction, the AnesQ of CT1 was significantly higher than MRI and CT2 with an effect size (95% CI) of 0.35 (0.14 – 0.56) and 1.05 (0.74 –

1.35); the AnesQ of the MRI was also significantly higher than CT2 with an effect size

(95% CI) of 0.70 (0.43 – 0.97). In addition, the AnesQ during the procedure was significantly higher in CT1 than CT2 with an effect size (95% CI) of 0.27 (0.02 – 0.52).

Related to the anesthetic phases of the CT1 and the MRI, the AnesQ after induction was significantly higher than during the procedure or the end with an effect size

(95% CI) of 1.05 (0.82 – 1.23) and 1.39 (1.08 – 1.70) for the CT1 and 0.82 (0.68 – 0.97) and 0.83 (0.63 – 1.02) for the MRI; during the procedure of the CT1 it was also significantly higher than the end with an effect size (95% CI) of 0.34 (0.05 – 0.64).

There was no significant difference for AnesQ between different anesthetic phases for

CT2.

The T1, T2, and T3 were not significantly different between treatment treatments or imaging modalities. The T4 was not significantly different between treatments but was significantly longer during CT1 with an effect size (95% CI) of 4.9 (2.9 – 7.0) to

MRI and 5.5 (2.5 -8.5) minutes to CT2. The T5 was significantly longer for A-S compared to P-S or P-M with effect sizes (95% CI) of 22.0 (10.6 – 33.4) and 18.0 (6.2 –

29.8) minutes, respectively. It was also significantly longer for A-M compared to P-S or

P-M with effect sizes (95% CI) of 21.6 (9.9 – 33.3) and 17.6 (5.7 – 29.5) minutes.

The recovery score data are presented in Table 5. The inter-rater agreement of

(MS) to (PL) and (AV) to (CM) was substantial (Landis & Koch 1977) with weighted kappa (95% CI) coefficient of 0.7988 (0.6870 – 0.9106); 0.8014 (0.6920 – 0.9107);

0.7010 (0.5645 – 0.8376), respectively. Hence only the ExtQ and RecQ directly scored

86 by a researcher (PL) were used. There was no difference between treatments regarding to

ExtQ or RecQ. The ExtQ immediately after extubation was significantly lower than the score at five and 10 minutes post-extubation with effect sizes (95% CI) of 0.41 (0.14 –

0.68) and 0.53 (0.21 – 0.85). The RecQ significantly decreased over 15, 30, 45 and 60 minutes post-extubation (p < 0.0001).

DISCUSSION

This study demonstrates that in fentanyl sedated dogs, co-induction with midazolam reduces the induction dose requirement and improves the induction quality for endotracheal intubation for both alfaxalone and propofol. The quality of induction was shown to be better with the inclusion of midazolam in the induction protocol, compared to the saline treatment, and the A-M treatment also scored better in comparison to P-S.

This induction quality score has the number of additional induction boluses required as part of the scoring, hence, Add-Dose not surprisingly varies in a manner similar to the

IndQ.

All dogs had a mild to moderate sedation with fentanyl, before administration of the induction drug. The investigators were not aware of the treatments allocation and the induction was always scored and performed by the same researcher to prevent bias and to make comparisons from this study more objective. In general, a smooth induction process with drugs that are safe and effective to allow endotracheal intubation is desired, especially in critically ill patients. Both P-M and A-M demonstrated the smoothest induction quality with a significant reduction in the total anesthetic dose of the induction drug. In addition, A-M also appeared superior to P-S. The real benefit of this dose reduction in healthy animals may merely be cost and ease, but in sick, compromised patients, the reduction in dose may potentially also equate to better cardiovascular stability although further studies are required to evaluate this. The actual mechanism by which benzodiazepines, as co-induction drugs, help promote a dose reduction of the induction anesthetic drug and allow for a smoother induction is still unclear. Synergism and/or additive effects with the primary induction anesthetic drug

88 are most likely. In vitro research shows that benzodiazepines potentiate both GABA and a variety of GABAA receptor allosteric agonists by inhibiting transitions from the open state to the longest-duration closed states (Li et al. 2013).

Benzodiazepine co-induction with propofol induction in dogs has shown mixed results regarding a dose reduction of propofol. The different results could be attributed to multiple factors, including sedation level of the dogs, which benzodiazepine is used, dose of the benzodiazepine, speed of injection of the benzodiazepine, sequence of administration with propofol, and initial dose and rate of propofol administration. One investigation in dogs reports a dose reduction of 36% of propofol when diazepam (0.4 mg kg-1, IV) was given before propofol but not with a lower diazepam dose (0.2 mg kg-1, IV)

(Ko et al. 2006); similarly others have not shown a dose reduction irrespective of the dose, sequence, or speed of administration (Ko et al. 2006; Braun et al. 2007; Robinson &

Borer-Weir 2013).

Midazolam has the potential to be more reliable in causing a reduction in the induction dose of propofol, related to the lower potency and lipid solubility characteristics of diazepam when compared to midazolam (Mohler & Okada 1977; Reves et al. 1978; Buhrer et al. 1990; Horikawa et al. 1990). We administered midazolam after an initial bolus of the primary induction anesthetic since the benefits on dose reduction are more reliable when administered in this order. Several studies have shown signs of excitement when midazolam is administered before propofol, which may interfere with the required total dose of propofol to achieve induction and intubation. In one study, midazolam, 0.25 mg kg-1, IV, administered over 30 seconds 2 minutes prior to propofol titrated to effect at a rate of 4 mg kg-1 min-1, IV, did not result in a dose reduction but

89 resulted in excitement in 95% of the dogs, despite intramuscular (IM) sedation with acepromazine (0.025 mg kg-1) and morphine (0.25 mg kg-1) 30 minutes before induction

(Covey-Crump & Murison 2008). In contrast, midazolam, 0.25 mg kg-1, IV, administered over 1 minute, and 30 seconds prior to propofol, titrated to effect at a rate of

2 mg kg-1 min-1, IV, resulted in a 47% dose reduction versus propofol alone (1.7 mg kg-1 versus 3.2 mg kg-1) but excitement was still noted in 45% of the dogs, despite prior IM sedation with acepromazine (0.02 mg kg-1) and morphine (0.4 mg kg-1) 30 minutes before induction (Sanchez et al. 2013). Similarly, this same dose of midazolam (0.25 mg kg-1,

IV) given over 15 seconds, immediately prior to propofol titrated to effect (3 mg kg-1 min-1), showed an 18% dose reduction versus propofol alone (2.8 versus 3.4 mg kg-1), despite signs of excitement in 55% of the dogs, despite prior IM sedation with acepromazine (0.025 mg kg-1) and morphine (0.25 mg kg-1) 30 minutes before induction

(Hopkins et al. 2013). Conversely, when midazolam (0.2 – 0.5 mg kg-1, IV) was administered after an initial bolus of propofol, 1 mg kg-1, IV, and propofol titrated to effect at rate of 2 - 4 mg kg-1 min-1, the incidence of excitement was less (12-18%) and there was a more consistent dose reduction (38-66%) (Robinson & Borer-Weir 2013;

Sanchez et al. 2013).

In this study, we chose to administer an initial IV bolus of alfaxalone of 0.5 mg kg-1 or propofol of 1 mg kg-1, which is lower than the average induction dose with or without midazolam co-induction used in dogs in other studies. This was done to ensure the results were able to demonstrate any potential dose reduction. Considering this lowered initial dose, two dogs each in A-S and A-M and one dog in P-M did not require any additional doses to allow endotracheal intubation, suggesting that we could have

90 potentially administered an even lower initial bolus dose. Nevertheless, the initial dose was chosen in our study to avoid potential excitement of furthered lower initial induction dosages and subsequent inability to demonstrate a potential dose reduction with midazolam co-induction. The fact that the mean number of additional doses was similar between A-S and P-S, and A-M and P-M indicates that the initial boluses were equivalent between the propofol and alfaxalone treatments.

The speed of injection of the induction agent is also an important factor that can influence the quality of induction. In sedated humans, the slower the administration of propofol during induction, the greater the effect on lowering the total dose required for loss of verbal contact (Stokes & Hutton 1991). Similarly, in sedated cats, the slower the administration of alfaxalone, the greater the effect on lowering the total dose required for endotracheal intubation (Bauquier et al. 2015). In the technical note provided by the pharmaceutical company, the induction dose of alfaxalone (2 -3 mg kg-1, IV) is recommended to be given as a slow infusion over 60 seconds or a quarter given every 15 seconds. In our study, the titration rate of propofol and alfaxalone were approximately

2.5 mg kg-1 min-1 and 1.25 mg kg-1 min-1, respectively. Our injection rate of propofol during induction was at the low end of the range used in other studies (2-4 mg kg-1 min-1)

(Covey-Crump & Murison 2008; (Hopkins et al. 2013; Robinson & Borer-Weir 2013;

Sanchez et al. 2013). Comparative results for alfaxalone and midazolam co-induction are not available.

A limitation of our study is that we did not standardize the speed of injection of the propofol or alfaxalone during the induction process with a syringe pump. However, we chose our methods to allow our results to predict the effects in a private practice

91 setting and not necessarily a specialty clinic or academic setting. We believe that our injection rate was slow enough to allow evaluation of depth and assessment of induction quality.

In our study, the midazolam co-induction provided a dose reduction of 25% for propofol and 37% for alfaxalone without any excitement in fentanyl sedated healthy research dogs. The percentage of dose reduction of propofol in our study is less than previous studies when midazolam was administered after a bolus of propofol (Robinson

& Borer-Weir 2013; Sanchez et al. 2013). This may due to the administration of fentanyl, which reduced the induction dose, overriding the impact of midazolam. In our study, mild to moderate sedation was noted from 7 ug kg -1, IV, of fentanyl without any clinical excitement. In dogs, peak concentrations of fentanyl are achieved in the brain tissue at 10-15 min after an IV injection of 12.5 ug kg -1 (Ainslie et al. 1979). There was a 17% reduction in the propofol induction dose when 2 µg kg-1, IV, fentanyl was given 2 minutes before propofol was titrated to effect at a rate of 4 mg kg-1 min1 (Covey-Crump

& Murison 2008). Moreover, the induction doses of the two control treatments, A-S and

P-S, were relatively lower than the doses of propofol or alfaxalone reported in the literature in pre-medicated dogs supporting a dose reduction effect of fentanyl in our study with the median time of fentanyl administration to the start of induction ranging from 9 to 14 minutes.

The TIVA rate of propofol alone used in this study (310 [274-351] µg kg-1 min-1) in healthy dogs was higher than the rate required in clinically non-sedated sick dogs undergoing an MRI (292 ± 119 µg kg-1 min-1) (Caines et al. 2014) and lower than in clinically healthy dogs sedated with subcutaneous acepromazine (0.01 mg kg-1) and

92 morphine (0.4 mg kg-1) undergoing surgery (370 ± 90 µg kg-1 min-1) (Suarez et al. 2012).

The TIVA rate of alfaxalone alone in our study (87 [77-98] µg kg-1 min-1) was higher than the dose required in healthy research dogs sedated with IV acepromazine (0.02 mg kg-1) and hydromorphone (0.05 mg kg-1) (70 µg kg-1 min-1) (Ambros et al. 2008) and lower than in clinically healthy dogs undergoing surgery (110 ± 10 µg kg-1 min-1) (Suarez et al. 2012).

Interestingly, in our study midazolam lowered the required TIVA dose of propofol

(268 [233-309] versus 310 [274-351]; µg kg-1 min-1) but not alfaxalone (83[74-94] versus

87 [77-98]; µg kg-1 min-1). Plasma concentrations of midazolam were not measured in this study, but would allow quantifying if differences exist on midazolam’s pharmacokinetics when administered with alfaxalone or propofol or if different drug interactions exist. Aside of the impact of midazolam, the TIVA rate was also significantly influenced by the diagnostic procedure of CT or MRI either after induction, during the procedure or at the end of the procedure. For the first CT, the dogs underwent an intervertebral disc injection of gelified ethanol for an unrelated study

{Mackenzie, 2016}. The dose rate for the first CT was higher irrespective of treatments during, and at the end of the procedure when compared to the second CT or the MRI

TIVA rates. This was to be expected and is likely related to the greater expected or real stimulation associated with the injection itself that, despite treatment blinding, resulted in dose adjustments from those monitoring the dogs for the injection. The TIVA dose was also increased over time in the first CT and MRI but was not significantly different during the second CT. This too is expected, and is related to the injection during the first CT or the scanning noise during the MRI. These periods of TIVA dosing

93 adjustments would not be expected to be impacted by the treatments themselves, as our results indicate, but by the phases of the anesthetic period.

The adjustments in rate and maintenance with either propofol or alfaxalone TIVA were readily achieved in all of our dogs. There was no difference in the ability to maintain TIVA for the CT or MRI, as indicated by similar AnesQ. However, as with the TIVA rate alteration, the AnesQ changes were related to the first or second CT or

MRI as well as the anesthetic event during the diagnostic procedure. A higher AnesQ, indicative of a lighter anesthetic plane, was present after induction versus when the dogs were undergoing the procedure or the end.

The time to extubation was approximately 6.4 – 7.1 minutes, which is shorter than the reported range of 10 – 15 minutes in previous studies (Ambros et al. 2008; Suarez et al. 2012). Our criteria for extubation may be the main reason contributing to this difference. In our study, extubation was initiated once a strong medial palpebral reflex was present in combination with the dog’s eye rolling up, but just prior to the swallow.

Other studies use the swallow reflex as an end-point. In our study, it was common, during the extubation process, for dogs to swallow in response to movement of the cuff of the endotracheal tube past the rima glottis, indicating that they were also ready for extubation, but rather than the swallow being spontaneous, it was induced. The purpose of this type of extubation was to minimize any chance of extubation-related excitement that may obscure the effects of the anesthetic on recovery parameters.

The recovery quality after propofol and alfaxalone TIVA in dogs has been compared and reported in dogs. Without pre-medication, dogs were reportedly sensitive to external stimulation or demonstrated adverse effects such as tremors, rigidity, or

94 myoclonus at recovery after propofol or alfaxalone induction (Morgan & Legge 1989;

Ferre et al. 2006; Maney et al. 2013). Other research (Ambros et al. 2008) and clinical investigations of TIVA for ovariohysterectomy (Suarez et al. 2012) in dogs did not find a significant difference between propofol and alfaxalone with regard to recovery times or quality, which was reported as good to excellent. However, both of these studies pre-medicated the dogs with drugs that are longer acting - acepromazine and morphine

(Suarez et al. 2012) or acepromazine and hydromorphone (Ambros et al. 2008) - compared to fentanyl, which was used in our study. In our study, RecQ was not different between propofol and alfaxalone with or without midazolam and was in general smooth with only mild excitement in some dogs. The mild excitement seen in some dogs in our study could have been minimized if longer-duration sedatives were used or additional sedatives were administered prior to recovery. However, our study design was to simulate the anesthetic protocol commonly used in critical canine patients for diagnostic procedures. It is possible that excitement may not be noted in clinical cases with other confounding factors such as a compromised health status.

Dogs in the alfaxalone treatments required a longer time to stand than dogs in the propofol treatments (65 minutes versus 45 minutes), despite no difference in the time to extubation between the treatments. The time to standing after alfaxalone TIVA in our study is similar to those from other studies, where it ranged from 52 -74 minutes (Ambros et al. 2008; Suarez et al. 2012); whereas the time to standing after propofol TIVA was shorter than the range of 70 – 90 minutes reported in other studies (Ambros et al. 2008;

Suarez et al. 2012). Comparisons with other studies should take into account the type of pre-medication, TIVA dose and time, and type of procedure performed. We handled

95 both treatments the same way and, therefore, it is not clear why the time to standing was different in the propofol treatments but not the alfaxalone treatments, compared to other studies.

In conclusion, midazolam is a suitable co-induction agent with either propofol or alfaxalone due to the reduced induction dose, improved induction quality and satisfactory recovery characteristics. Improvement of induction quality and primary injectable dose reduction could also benefit clinical case management. However, further research is warranted to investigate the effects of midazolam co-induction in critical cases.

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Figure 1. Study timeline with administration of fentanyl (F); propofol (P) or alfaxalone (A) 1 with either saline (S) or midazolam (M); initiation (//) and discontinuation (//) of total 2 intravenous anesthesia (TIVA) is indicated. The arrows between phases indicate transfer of 3 dogs. Times are as follows; time from sedation to TIVA discontinuation: T1; time from 4 sedation to induction: T2; time from induction to TIVA discontinuation: T3; time from TIVA 5 discontinuation to extubation: T4; time from TIVA discontinuation to dogs standing: T5. Dogs 6 were scored at time of endotracheal extubation, 0, as well as 5 and 10 min after. Additional 7 recovery scoring continued for 45 minutes at time points 15, 30, 45 and 60 min post-extubation. 8

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Table 3. Descriptive quality scores for Sedation (SedQ) (0-none to 3-profound), time from 15 premedication to induction (T2), induction quality (IndQ) (0-smooth to 4-not possible), number 16 of additional doses during anesthetic induction (Add-Dose), and total anesthetic dose (TotalD). 17

All dogs were randomly assigned to one of the 4 treatments; Propofol (P) + Midazolam (M) 18

(P-M); Alfaxalone (A) + M: (A-M); P + saline (S) (P-S); and A + S (A-S). Treatments were as 19 follows: Fentanyl (F) (7 µg kg-1, IV) was administered, followed by an initial IV bolus of P (1 20 mg kg-1) or A (0.5 mg kg-1) 10 min after F. Either M (0.3 mg kg-1, IV) or S was administered 21 immediately after the initial P or A bolus followed by additional boluses of the respective 22 induction drug every 6 seconds (Add-Dose) to allow endo-tracheal intubation. 23

24 PS PM AS AM SedQ 1.9 (1.2 - 2.5) 2.4 (1.7 - 3.1) 1.7 (1.0 - 2.3) 2.0 (1.3 - 2.6) 25 (0 - 3) T2 9.0 (6.4 - 12.7) 14.2 (9.9 - 20.2) 13.0 (9.3 - 18.1) 12.8 (9.1 - 18.1) 26 (min) 27 IndQ 2.3 (1.8 - 2.9)a 0.9 (0.4 - 1.5)bc 1.6 (1.1 - 2.1)ac 0.7 (0.1 - 1.2)b 28 (0 - 4) Add-Dose 0.9 (-0.3 – 29 3.9 (2.8 – 5.1)ac 2.1 (0.9 – 3.3)ad 2.8 (1.7 – 4.0)a (number) 2.1)bd 30 TotalD 0.62 (0.4 - 2.1 (1.8 - 2.3)*a 1.5 (1.3 - 1.8)*b 0.98 (0.7 - 1.2)#c 31 (mg kg-1) 0.9)#d 32 Data are presented as mean with 95% confidence interval, except for (T2) presented as median 33 with 95% confidence interval. Difference superscript letters indicates statistical difference 34 between the treatments. TotalD was not compared between P and A. Symbols are as follows for 35 treatment comparisons PS vs. PM (*) and AS vs AM (#). 36

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Table 4. Total intravenous anesthesia doses (TIVA), time from administration of sedation to 37

TIVA discontinuation (T1), total anesthesia time from the start of induction to TIVA 38 discontinuation (T3), time from TIVA discontinuation to extubation (T4), time from TIVA 39 discontinuation to standing (T5), and simple descriptive anesthesia maintenance quality scores 40

(AnesQ) (0-no adjustments to 4-intervention required) following induction with either propofol 41

(P) + Midazolam (M) (PM); Alfaxalone (A) + M: (AM); P + saline (S) (PS); and A + S (AS). 42

(see Table 3 for key). 43

PS PM AS AM 44 a b TIVA rate 310 268 87 83 45 µg kg-1 min (274 - 351) (233 - 309) (77-98) (74 - 94) 46 T1 105.0 110.4 105.9 107.5 (min) (114.0 - 96.0) (119.6 - 101.2) (114.6 - 97.3) (116.5 - 98.4) 47 T3 94.4 89.5 88.9 93.1 48 (min) (85.7 - 104.1) (80.8 - 99.1) (81.6 - 96.9) (84.7 - 102.2) 49 T4 6.4 6.6 7.7 7.1 (min) (4.1 – 8.7) (4.3 – 8.9) (5.7 – 9.6) (4.9 – 9.3) 50 T5 43.8 47.8 65.9 65.4 51 (min) (53.8 - 33.8)a (58.3 - 37.3)a (75.7 – 56.0)b (75.6 – 55.2)b 52 AnesQ 0.48 0.35 0.43 0.40 (0-4) (0.32 – 0.64) (0.18 – 0.52) (0.29 – 0.57) (0.25 – 0.56) 53 54 Data are presented as mean with 95% confidence interval except for TIVA rate presented as 55 median with 95% confidence interval. Difference superscript letters indicates statistical 56 difference between the treatments. TIVA was not compared between P and A. 57

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Table 5. Descriptive quality scores of extubation (ExtQ) (0-very calm to 4- extreme 58 excitement) and recovery (RecQ) (0-excitable to 3-profound sedation) after extubation following 59 propofol (P) or alfaxalone (A) total intravenous anesthesia for diagnostic imaging. Each dog 60 was randomly assigned to induction agents as follows: P + Midazolam (M) (PM); A + M: (AM); 61

P + saline (S) (PS); and A + S (AS). The ExtQ was assessed immediately, 0, and 5 and 10 62 minutes after extubation. The RecQ was assessed 15, 30, 45 and 60 minutes after extubation. 63

Time after extubation PS PM AS AM (min) 0.0 0.3 0.5 0.1 0 (-0.7 – 0.6) (-0.4 – 1.0) (-0.2 – 1.1) (-0.5 – 0.8) 0.4 0.8 0.6 0.7 ExtQ 5 (-0.2 – 1.0) (0.1 – 1.4) (0.0 – 1.3) (0.1 – 1.4) 0.6 0.3 0.9 1.2 10 (0.0 – 1.2) (-0.4 – 1.0) (0.3 – 1.5) (0.5 – 1.8) 2.1 2.5 2.6 2.7 15 (1.4 – 7.8) (1.8 – 3.2) (1.9 – 3.2) (2.0 – 3.3) 1.2 1.7 2.3 2.3 30 (0.6 – 1.9) (1.0 – 2.4) (1.7 – 3.0) (1.7 – 3.0) RecQ 0.3 0.8 1.2 1.3 45 (-0.3 – 1.0) (0.1 – 1.5) (0.6 – 1.8) (0.7 – 2.0) 0.1 0.2 0.4 0.6 60 (-0.6 – 0.8) (-0.4 – 1.0) (-0.2 – 1.1) (-0.1 – 1.3) 64 Data are presented as mean with 95% confidence interval. Difference superscript letters indicate 65 statistical difference between the treatments. 66

67 68

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Appendix 1. Simple descriptive quality scores for sedation prior to anesthetic induction (SedQ) 69 in dogs, 10 minutes after administration of fentanyl, 7 µg kg-1, IV. (modified from Caines et al. 70

2014) 71

72 Sedation Quality Score

0 Bright and alert - no sedation and/or excitable- dysphoric (excited, anxious, difficult

to restraint in lateral recumbency, very interactive and responsive, vocalizing, very

reactive to noise or touch

1 Calm - minimal sedation, quiet but still alert and aware of surroundings, mild

resistance to restraint in lateral recumbency, moderate response to noise or touch

2 Mild to moderate sedation - quiet, relaxed, minimal restraint required in lateral

recumbency, mild response to noise or touch

3 Profound sedation - quiet, very relaxed, no restraint necessary in lateral

recumbency, no response to noise or touch

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Appendix 2. Simple descriptive quality scale for induction and intubation (IndQ) with 73 propofol or alfaxalone with or without midazolam co-induction after sedation with fentanyl in 74 dogs. 75

Induction and Intubation Quality Score 0 Smooth with no Resistance - Dog relaxes within 30 seconds, no jaw tone, no lateral palpebral, no tongue tone, no response to laryngoscope placement. Dog easily intubated with the initial bolus dose within 45 seconds. 1 Slight Resistance but Smooth - Dog relaxes within 30 seconds, no jaw tone, no lateral palpebral, no tongue tone, no response to laryngoscope placement. However, dog does cough on intubation and/or swallows once intubated. Requires 1-2 additional subsequent boluses of the induction drug. Dog is intubated within 45 seconds. 2 Mild-Moderate Resistance - Dog relaxes within 30 seconds, no jaw tone, no lateral palpebral. However, dog responds to laryngoscope placement with tongue curl. Requires 1-2 additional subsequent boluses of the induction drug to proceed. Cough and or swallow may also be noted. Dog is intubated within 60 seconds. 3 Moderate Resistance – Unacceptable Quality - Dog does not relax initially within 30 seconds and requires 2-3 subsequent injectable boluses of the induction drug to proceed to intubation. Resistance to intubation attempt within 45 seconds (cough, tongue curl, and or other movement) requiring subsequent additional doses during the intubation process. Dog relaxes after intubation without further movement but is at a light plane. Dog is intubated within 60 seconds. 4 Excitement - Paddling, hyperkinesis, vocalizing, defecation, urination. Unable to intubate without significant number of subsequent doses of the induction drug. Intubation takes more than 60 seconds. 76 77

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Appendix 3. Simple descriptive quality scores for anesthetic maintenance (AnesQ) with 78 propofol or alfaxalone TIVA with or without midazolam co-induction after sedation with 79 fentanyl, during diagnostic CT or MRI in dogs. (modified from Caines et al. 2014) 80

Anesthetic Maintenance Quality Score 0 No adjustments required in the TIVA rate of propofol or alfaxalone during transfer or maintenance in MRI/CT. Relaxed jaw tone, slight to no medial palpebral response, muscle relaxation during positioning or procedures. 1 One additional bolus of propofol or alfaxalone and an increase in the TIVA rate during transfer or maintenance in MRI/CT. Dog appears light with strong jaw tone and medial palpebral during depth assessment, but no doses are required. No movement is noted. Change in anesthetic depth happens only once. 2 One to two additional boluses of propofol or alfaxalone and an increase in the TIVA rate during transfer or maintenance in MRI/CT. Signs noted include slight increases in HR, BP, bucking ventilator, increased jaw tone, strong medial palpebral reflexes during the MRI or disc injection, muscle tone, and rigidity. Change in anesthetic depth occurs 2-3 times but is easily controlled. 3 Two to three additional boluses of propofol or alfaxalone with increases in the TIVA rate during transfer or maintenance in MRI/CT. Signs noted include slight increases in HR, BP, bucking ventilator, increased jaw tone, strong medial palpebral reflexes during the MRI or disc injection, periodic movement. Change in anesthetic depth occurs 2-3 times or more and is not easily controlled. 4 Four plus additional boluses of propofol or alfaxalone with increases in the TIVA rate during transfer or maintenance in MRI/CT. Signs noted include dramatic changes in HR, BP, bucking ventilator, strong jaw tone, movement, brisk palpebral, and overall increased responsiveness during the MRI or disc injection.

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Appendix 4. Simple descriptive quality scores for extubation (ExtQ) immediately after 82 extubation and 5 and 10 minutes after extubation in dogs recovering from propofol or alfaxalone 83

TIVA with or without midazolam co-induction after sedation with fentanyl, during diagnostic 84

CT or MRI in dogs. (modified from Caines et al. 2014) 85

86 Extubation Quality Score 87 0 Very calm, smooth, no excitement. No paddling, vocalization or trembling 88 89 1 Smooth, with slight short excitement of < 30 seconds. No paddling, vocalization 90 or trembling 91 2 Moderately smooth with mild excitement. Some paddling, vocalization and 92

trembling.

3 Not smooth and with excitement. Paddling, vocalization and trembling. Vomiting

may be observed.

4 Extreme excitement. Paddling, vocalization and/or aggression. Vomiting may be

observed. Convulsions may be observed. Sedation/therapy required.

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Appendix 5. Simple descriptive quality scores for recovery (RecQ) 15, 30, 45 and 60 minutes 93 after extubation in dogs recovering from propofol or alfaxalone TIVA with or without 94 midazolam co-induction after sedation with fentanyl, during diagnostic CT or MRI in dogs. 95

(modified from Caines et al. 2014) 96

Recovery Quality Score

0 Excitable with no apparent sedation or depression (excited, ambulatory, difficult

to restraint in lateral at recovery for transport or in cage, very interactive,

responds to voice and caregivers, attempting to go sternal within 15 minutes of

extubation and/or ambulatory after 30 min. Animal is bright and looks similar to

their status prior to anesthesia).

1 Calm with no apparent sedation or depression (alert, mild to moderate resistance

to restraint in lateral for transport or in cage, moderately interactive, responds to

voice and caregivers, no attempts to sternal recumbency after 15-30 minutes

and/or not ambulatory after 30-60 minutes).

2 Apparent sedation/depression (quiet, minimal restraint required to keep animal in

lateral recumbency for transport or in cage, mild response to voice or touch – no

attempts to sternal recumbency and/or non ambulatory after 1 hour).

3 Profound sedation/depression (dull, minimal to no restraint required to keep

animal in lateral recumbency for transport or in cage, does not respond to voice

or touch, - no attempts to sternal recumbency and/or non-ambulatory for >1 hour

post recovery).

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CHAPTER III

CARDIO-PULMONARY EFFECTS OF PROPOFOL OR ALFAXALONE WITH OR

WITHOUT MIDAZOLAM CO-INDUCTION FOLLOWED BY TOTAL

INTRAVENOUS ANESTHESIA IN FENTANYL SEDATED DOGS

SUMMARY

Objective To compare cardio-pulmonary function between propofol (P) and alfaxalone (A) with or without midazolam (M) during induction followed by total intravenous anesthesia

(TIVA) for diagnostic imaging in fentanyl sedated dogs.

Experimental design Prospective, randomized, incomplete Latin-square crossover, blinded trial.

Animals Ten research dogs weighing a mean (SD) of 24.5 (3.1) kg.

Methods Dogs were randomly assigned to P with saline (S), A-S, P-M, and A-M. Fentanyl (7

µg kg-1, IV) was administered 10 minutes prior to an IV bolus of P (1 mg kg-1) or A (0.5 mg kg-1) followed by M (0.3 mg kg-1, IV) or S and additional boluses of P or A for intubation, followed by maintenance with P or A TIVA. Heart rate (HR), systolic (SAP), mean (MAP) and diastolic (DAP) blood pressure, cardiac index (CI), respiratory rate (fR), end-tidal carbon dioxide partial pressure (P’CO2) and arterial blood gas analysis were compared. Analysis included a general linear mixed model with post-hoc analysis (p < 0.05).

Results After induction, HR was higher in A-M than A-S (p = 0.049) and P-S (p < 0.001).

During imaging, HR of A-S (p = 0.012) and A-M (p = 0.021) were higher than P-S. Before recovery, HR of A-M was higher than P-S (p = 0.009). The overall SAP of A-S was significantly higher than A-M (p < 0.001) and P-M (p = 0.001). There was no significant treatment difference for MAP, DAP, CI, fR, prevalence of apnea, PE’CO2, and blood gas

111 values. However, CI and HR significantly decreased at the end of imaging compared to other phases (p < 0.001).

Conclusions and clinical relevance

There is no significant cardio-pulmonary difference between treatment despite different P or

A dosing. The decrease in CI and HR at the end warrants close monitoring.

Keywords propofol, alfaxalone, midazolam, dog, TIVA

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INTRODUCTION

In veterinary anesthesia, co-induction agents may be used during the induction period principally to reduce the dose of the primary induction agent and ensure a smooth overall endotracheal intubation process. Co-induction with benzodiazepines has been most extensively studied with propofol induction in dogs. Midazolam shows the most consistent dose-reduction results especially when given after the initial bolus of propofol however the cardio-pulmonary benefit has not been clearly defined (Robinson & Borer-Weir 2013;

Sanchez et al. 2013).

The cardio-pulmonary depression produced with propofol and alfaxalone induction is generally dose-dependent (Ismail et al. 1992; Puttick et al. 1992; Muir & Gadawski 1998;

Muir et al. 2008; Keates & Whittem 2012). A potential goal associated with the dose reduction of propofol or alfaxalone provided by co-induction agents is to minimize the negative cardio-pulmonary effects. However, the benefit of combining the primary injectable anesthetic with a co-induction agent on the overall cardiovascular performance, and incidence of hypoventilation or apnea have not been clearly defined, and their combined use remains controversial in both human and veterinary anesthesia (Anderson & Robb 1998;

Jones et al. 2002; Goel et al. 2008; Hopkins et al. 2013). In addition, most studies looking at the effects of co-induction agents on primary induction anesthetics have been completed using healthy patients and little is known of the combined effect in critical patients. The scientific literature has primarily focused on co-induction investigations with propofol to date, with minimal information on alfaxalone.

Total intravenous anesthesia (TIVA) has some advantages over inhalant anesthesia for maintenance, especially during advanced imaging, including reduced equipment need and potential workplace hazards from inhalant exposure to personnel. Propofol has the ideal

113 pharmacokinetic profile for TIVA use (Waelbers et al. 2009) and was demonstrated to have better cardiovascular function when compared to isoflurane in research dogs (Keegan &

Greene 1993; Deryck et al. 1996; Iizuka et al. 2013) and in clinical dogs with intracranial disease undergoing magnetic resonance imaging (MRI) (Caines et al. 2014). Alfaxalone has been remarketed for almost a decade and has shown similar anesthetic effects to propofol in dogs (Ambros et al. 2008; Suarez et al. 2012; Maney et al. 2013). However, detailed comparisons between propofol and alfaxalone TIVA for diagnostic imaging are lacking.

To the authors’ knowledge, scientific studies investigating cardio-pulmonary measurements, such as direct systolic (SAP), mean (MAP) and diastolic (DAP) arterial blood pressure and cardiac output (CO), during co-induction with midazolam and either propofol or alfaxalone followed by TIVA are not available, but could offer clarification of the cardio-pulmonary benefit of their combination. The objective of this research is to compare cardio-pulmonary function between propofol and alfaxalone with or without the co-administration of midazolam during anesthetic induction followed by TIVA for computer tomography (CT) or magnetic resonance imaging (MRI) in fentanyl sedated research dogs.

We hypothesize that in healthy ASA I dogs there will be no difference in cardio-pulmonary effects during the anesthetic induction with propofol or alfaxalone with or without midazolam nor differences during TIVA maintenance.

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MATERIALS AND METHODS

Animals

All procedures were approved by the Animal Care Committee, University of Guelph, and followed Canadian Council on Animal Care Guidelines. Ten healthy research crossbred hound dogs, mean (range) age 3.4 (1.9 – 5.5) years, mean (SD) weight 24.5 (3.1) kg, ASA classification I were used. Health status was based on a general physical examination, complete blood count and biochemistry panel. Dogs were included in a simultaneous aligned study evaluating the dose reduction and anesthesia quality of alfaxalone or propofol during induction and TIVA as well as recovery characteristics with or without midazolam detailed in the anesthetic methods below.

Study Design

This study was a prospective, blinded, randomized, incomplete Latin-square crossover research trial with at least a seven days washout between 5 separate anesthetic events. Randomization was performed using a computer software program (GraphPad,

California, USA) to ensure blinded allocation of dogs between the treatments. Papers containing the anesthetic treatment allocation of dog for the research day were organized and opened by anesthesia research technicians. The research technicians also prepared the syringes for the randomized drugs for induction, additional doses, constant rate infusions, as well as adjustment of TIVA doses. Drapes were used to cover the syringes and infusions lines to prevent either investigator (MS; PL) from identifying the drug by color during induction dose assessments and collection of cardio-pulmonary data.

Dogs underwent MRI (first, third and fourth anesthetic event), a CT guided intervertebral disc injection of gelified ethanol (CT1; second event) in a parallel but unrelated

115 study (Mackenzie et al. 2016) and computed tomography without injection (CT2; fifth event) while maintained on propofol or alfaxalone TIVA. The results of the induction dose and quality, TIVA dose and maintenance and recovery characteristics are available elsewhere

(Liao et al. 2015a; Liao et al. 2015b).

Anesthesia (see Figure 2)

Preparation and instrumentation

The dogs were fasted for at least 12 hours but given free access to water prior to general anesthesia. Topical local anesthetic cream (Maxilene, RGR Pharma Ltd, Canada) was applied pre-emptively to the clipped area over the cephalic vein and dorsal pedal artery and the sites bandaged. Ten minutes later, the bandage over the cephalic vein site was removed and a 20-gauge catheter (Insyte-W; Becton Dickinson Infusion Therapy Systems, Utah, USA) was inserted and 0.1 mg kg-1 of meloxicam (Metacam®, Boehringer Ingelheim Canada Ltd,

Canada) administered intravenously (IV) prior to anesthesia. Thirty minutes after IV catheter placement, dogs were manually restrained in lateral recumbency, the bandage over the dorsal pedal artery removed and lidocaine (Lidocaine, Alveda Pharmaceuticals, Canada) (2%, 0.5 mL) infiltrated subcutaneously to facilitate the catheterization of the artery with a 20- or

22-gauge catheter.

Monitoring

Continuous ECG display (lead II), pulse-oximetry (SpO2), SAP, MAP and DAP, and capnography, which provided respiratory rate (fR) and end-tidal carbon dioxide partial pressure (PE´CO2), were monitored in the preparation area with a multi-channel monitor

(PM-9000 Express, Mindray Medical International Ltd, China). The fR was manually counted over 30 seconds in the awake dog. Another multichannel monitor (CC5; Cardiocap/5, GE

116

Datex Ohmeda, Canada) was used to monitor the same variables except ECG and SpO2, while the animals were in the magnetic resonance room. The SpO2 was monitored by a separate magnetic compatible pulse-oximeter (Nonin 8600v, Nonin Medical Inc, Minnesota,

USA). Similarly, in the CT room, variables were monitored with a CC5 or PM-9000 monitor, depending on the availability of the equipment.

The arterial catheter was connected by non-compliant extension tubing (Arterial

Pressure Tubing, ICU Medical Inc, Califonia, USA) to a pressure transducer (Transducer set;

Becton Dickinson Critical Care System Pte, Ltd, Singapore) and the multi-channel monitor for determination of SAP, MAP and DAP, and pulse contour cardiac output measurement

(PulseCO hemodynamic monitor, LiDCO Ltd, UK). With the dog in lateral recumbency, the level of the right atrium was assumed to be at the level of the manubrium and used as the zero reference for all blood pressure determinations. The equipment used for blood pressure measurement was assessed for accuracy at 100 mmHg against a mercury manometer

(Mercurial Sphygmomanometer, Japan) at the start of each study day. The infrared airway gas monitor was calibrated daily with gases (Anesthesia calibration gases, Scott Medical

Products, Pennsylvania, USA) specific for the monitor and attached to a line sampling at the mouth end of the endotracheal tube.

Arterial blood was sampled from the dorsal pedal arterial catheter less than 5 minutes before every lithium cardiac output measurement (LiDCO plus Hemodynamic Monitor,

LiDCO Ltd, UK) and analyzed immediately for arterial partial pressures of oxygen (PaO2) and carbon dioxide (PaCO2), pH, hemoglobin (Hb), lactate (Lac) and electrolyte concentrations including sodium (Na+), potassium (K+) and chloride (Cl-) (ABL 750;

Radiometer, Denmark). The remaining arterial blood was placed on ice and used to determine hematocrit and total proteins on the same day. Rectal temperature (Temp; 0C) was measured at each blood gas determination.

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Cardiac output measurement

The CC5 monitor was connected to the LiDCO to allow for PulseCO determinations using a 1-volt analog signal for 100 mm Hg pressure. With each LiDCO determination the

PulseCO monitor was calibrated with the LiDCO. Steps for preparing the lithium sensors

(Flow through Cell Electrode Assembly, CM10, LiDCO Ltd, UK), checking for suitable sensor voltage and stable baseline signal were as described in the operation manual (LiDCO

Plus Hemodynamic Monitor User’s Manual 1.0, LiDCO Ltd, UK). For LiDCO determinations, lithium chloride (6 µmol kg-1) (Lithium Chloride Injection, LiDCO Ltd, UK) was injected rapidly into the cephalic vein catheter 6 seconds after starting the injection phase on the LiDCO computer and simultaneously arterial blood was withdrawn by a peristaltic pump (LiDCO Flow Regulator; LiDCO Ltd, UK) into a disposable blood collection bag

(Disposable blood collection bag and tube; LiDCO) at a flow of 4 mL minute-1 across the lithium sensor. Cardiac index, stroke volume index and systemic vascular resistance index were calculated from the CO measured by LiDCO (CIL, SVIL, SVRIL) and cardiac index from the PulseCO (CIP) using the formulas below and assuming a central venous pressure

(CVP) of 5 mmHg.

Cardiac index (mL kg-1 min-1) = CO (mL min-1) ÷ body weight (kg)

Stroke volume index (mL kg-1 beat-1) = CI ÷ HR

Systemic vascular resistance index (mmHg mL-1 min-1 kg-1) = (MAP – CVP) ÷ CI

Anesthesia

With the dogs in lateral recumbency and attached to the multichannel monitor for continuous measurement of HR, SAP, MAP and DAP, PulseCO, and ECG, fentanyl (7 µg kg-1, IV) was administered (Fentanyl 50 µg mL-1, Sandoz Canada Inc, Canada). Dogs were randomly assigned to one of 4 treatments for anesthetic induction with propofol (Propofol 10

118 mg mL-1, Pharmascience Inc, Canada) or alfaxalone (Alfaxan® 10 mg mL-1, Jurox Pty

Limited, Australia) with or without midazolam (Midazolam 5 mg mL-1, Pharmaceutical

Partners of Canada Inc, Canada) or saline (0.9% Sodium Chloride, Hospira, Canada) and maintained by TIVA with respective injectable agent as follows; Treatment P-M: propofol (1 mg kg-1, IV) and midazolam (0.3 mg kg-1, IV); Treatment A-M: alfaxalone (0.5 mg kg-1, IV) and midazolam (0.3 mg kg-1, IV); Treatment PS: propofol (1mg kg-1 IV) and an equal volume of saline (0.06 mL kg-1, IV); Treatment A-S: alfaxalone (0.5 mg kg-1 IV) and an equal volume of saline (0.06 mL kg-1 IV). Initial doses of propofol or alfaxalone were administered as a bolus, flushed with 2 mL of saline IV and then the midazolam or an equal volume of saline was immediately administered. An additional 25% of bolus dose of propofol or alfaxalone was administered every 6 seconds upon request until successful endotracheal intubation could be performed (MS). No attempts to intubate were made until confirmed loss of lateral palpebral reflex and then relaxation of jaw tone occured.

Once intubated, dogs were maintained on TIVA with the same induction anesthetic, administered via syringe pump (Medfusion 3500, Smiths Medical Canada, Canada; Graseby

3500 Anesthesia Pump; Smiths Medical International Ltd, UK) at the following initial rates:

250 µg kg-1 minute-1 of propofol or 70 µg kg-1 minute-1of alfaxalone. Dogs were connected to an anesthetic machine with a rebreathing circuit (Universal F-Circuit; Dispomed, Canada) using an oxygen flow of 1.5-2.5 L minute-1. A manual breath was administered if the dog did not spontaneously ventilate for longer than 30 seconds in the first 15 minutes after induction.

-1 Thereafter, mechanical ventilation was initiated with a tidal volume of 10 mL kg and fR adjusted to PE´CO2 between 35 and 45 mmHg.

For the MRI, earplugs were placed in the ears and the dogs were disconnected from the anesthesia machine and monitor, transferred into the magnetic bore and positioned in dorsal recumbency. In the MRI acquisition room, the dogs were connected to another

119 anesthesia machine and monitor, both non-MRI compatible and therefore situated next to the control room workstation; the anesthetic machine was previously set up with breathing hoses that reached from the patient to the machine, and the monitor set up with extension tubing that reach the arterial catheter (944 cm). Intermittent depth assessments between the scan sequences were done by an anesthetist unaware of treatment allocation (PL).

For the CT scan, the dogs were transferred to the CT acquisition room with the induction anesthetic machine and PM-9000 monitor or switched to a separate anesthetic workstation and the CC5 monitor depending on the availability of anesthetic equipment. The dogs were positioned in the gantry in sternal recumbency. Depth of anesthesia was evaluated by an anesthetist unaware of treatment allocation (PL).

Adjustments to the TIVA rate, based on physical signs and physiological variables, were made every 5 minutes to maintain a light plane of anesthesia. Signs of inadequate depth included movement, brisk palpebral reflex, increased jaw tone, spontaneous blinking, or continual increases in HR, fR or SAP, MAP and DAP. When inadequate depth was present,

TIVA rates were adjusted by 25 µg kg-1 minute-1 for propofol and 7 µg kg-1 minute-1 for alfaxalone for minor depth change requirements. When a rapid increase in depth was warranted, top-up doses of propofol (0.25 mg kg-1) or alfaxalone (0.125 mg kg-1) were administered. Anesthetic administration rate was decreased when a progressive decrease in arterial pressure was noted, without respiratory fluctuations or attempts to ventilate spontaneously during mechanical ventilation, in addition to lack of palpebral reflexes and jaw tone. Hypotension was defined as MAP < 60 mmHg for more than 5 minutes. Hypotension was treated with an infusion of dopamine (2–10 µg kg-1 minute-1) at the anesthetist’s discretion if depth adjustment was not sufficient to reestablish normotension.

120

Cardio-pulmonary measurements

Cardio-pulmonary measurements were recorded in 4 different phases: 1) before and after fentanyl sedation (SED), 2) immediately after induction and for 15 minutes during spontaneous ventilation followed by commencement of intermittent positive pressure ventilation before the imaging procedure (IND), 3) during the CT or MRI imaging (PROC), and 4) after imaging prior to recovery from anesthesia (END). Following instrumentation the following phase measurements were recorded: SED: HR, fR and SAP, MAP, and DAP were recorded before (S-30), and 4 minutes after fentanyl (S-34); IND: HR, fR, SAP, MAP, DAP,

SpO2, and PE´CO2 were recorded immediately after endotracheal intubation (I-0) and every 5 min for 15 min (I-5, I-10, I-15) with spontaneous ventilation in lateral recumbency and then at 20 minutes after IPPV started (I-20) and prior to PROC; PROC: HR, fR, SAP, MAP, DAP,

SpO2, and PE´CO2 were performed every 5 minutes (P-0 to P-55); END: HR, fR, SAP, MAP,

DAP, SpO2, and PE´CO2 were performed once the dog was back to the induction area (E-0) and 5 minutes later (E-5). LiDCO was measured at S-34, I-15 and E-5, and PulseCO was measured during IND, and END, but not during PROC.

Statistical analysis

Statistical analyses were performed with standard computer software (SAS OnlineDoc

9.2; SAS Institute Inc., North Carolina, USA). Normality of data was tested using

Shapiro-Wilk, Kolmogorov-Smirnov, Cramer-von Mises, and Anderson-Darling tests (Proc

UNIVARIATE, SAS). Continuous data that were not normally distributed were log transformed for analysis, unless log transformation provided no improvement to distribution.

A general linear mixed model was used to model the results using Proc MIXED. Fixed effects for HR, SAP, MAP, DAP, fR, and PE’CO2 were treatment, imaging procedure, phase and time points; for CI, SVI, SVRI, temperature, and arterial blood gas analysis they were

121 treatment, imaging procedure and phases, including up to three-way interactions. Phases and time points were as outlined in the methods for analysis. Dog was considered a random effect and treated as a blocking variable. To model for the effects of repeated measures over time for HR, SAP, MAP, DAP, fR, and PE’CO2 on each dog, due to treatment and within a specific event of anesthesia, the following correlation structures, offered by SAS, were attempted: ar(1), arh(1), sp(pow)(time), toep, banded toep, toeph, banded toeph, and un and banded un.

The error structure, among those that converged, was chosen based upon the lowest Akaike

Information Criteria. Terms in the model were removed if non-significant, but preserving model hierarchy. Appropriate adjustments (Tukey or Dunnet test) for multiple comparisons were used. The chi square test was used to test if significant associations in the proportion of dopamine infusions between imaging modalities and treatments. Significance was set at p <

0.05. Residuals were examined to assess the ANOVA assumptions and plotted against the predicted values and the explanatory variables used in the model. Such analyses help reveal outliers, unequal variance, the need for data transformation, and other issues that need addressing.

The results are presented with adjusted mean if data are normally distributed or ordinal and adjusted median if not normally distributed based on a log transformation with

95% confidence interval (CI). The adjustment was done by applying standard ANOVA least-squares methods to estimate and test the results as if the data were in a complete

Latin-square, crossover design. When comparing between treatments, for those normally distributed continuous data and ordinal data, the effect size is provided as the difference between treatments with 95% CI. The p-value is provided for those not normally distributed and log transformed.

122

RESULTS

- Data for all the cardio-pulmonary variables, TIVA rate, fR, Hb, PaO2, and HCO3 were not normally distributed. All dogs recovered without complications from anesthesia. The

PROC duration ranged from 35-55 min and was not significantly different between treatments and data during this phase are reported for up to 40 minutes only.

The mean (95% CI) induction dose for the 4 treatments during cardio-pulmonary measurements was: P-S 2.1 mg kg-1 (1.8 - 2.3); P-M 1.5 mg kg-1 (1.3 - 1.8); A-S 0.98 mg kg-1

(0.7 - 1.2); and A-M 0.62 mg kg-1 (0.4 - 0.9). The overall median TIVA rate (95% CI) of the

P-S, P-M, A-S and A-M were 310 µg kg-1 min-1 (274 - 351), 268 µg kg-1 min-1 (233 - 309), 87

µg kg-1 min-1 (77 - 98), and 83 µg kg-1 min-1 (74 - 94), respectively. Detailed induction and

TIVA dosing results are presented elsewhere (Liao et al. 2015a; Liao et al. 2015b).

The HR, SAP, MAP and DAP are presented in Figure 3 and Table 6. Heart rate decreased after fentanyl administration in all treatments (phase SED), although this change was not significant. For the P-M treatment, HR during SED was higher than PROC (p =

0.003). During IND, HR for A-M was significantly higher than A-S (p = 0.049) and P-S (p <

0.001). During PROC, HR for A-S (p = 0.012) and A-M (p = 0.021) were higher than P-S.

During END, HR for A-M was higher than P-S (p = 0.009). For specific times during IND, there were no differences between treatments; HR decreased significantly in all treatments at

I-5 with respect to I-0 (p = 0.037), I-15 (p = 0.006), and I-20 (p < 0.001). The HR was also significantly higher at I-20 than at I-0 (p = 0.048). The HR at END was significantly lower than for all other phases (p < 0.001).

The HR was significantly higher during the CT1 for A-S (p = 0.028), A-M (p =

0.028), and P-M (p = 0.028) than for P-S; during the CT2, HR in the A-M treatment was significantly higher than A-S (p = 0.012) and P-S (p = 0.001); and during MRI there were no

123 differences between treatments. Comparisons between imaging modalities within treatments resulted in higher HR for A-S during CT1 than for CT2 (p = 0.013) and MRI (p = 0.042), while for A-M the HR during CT2 was higher than for MRI (p = 0.006).

Systolic arterial pressure in the A-S treatment was significantly higher than A-M (p <

0.001) and P-M (p = 0.001). The SAP of all treatments at S-34 was significantly higher than other time points (p < 0.001). During induction, the SAP of all treatments at I-0 was significantly higher than I-5 to I-20 (p < 0.001), and I-15 was also higher than I-20 (p =

0.001). During CT2, the SAP of all treatments at IND was significantly higher than PROC (p

= 0.002). The SAP of all treatments at P-35 (p = 0.001) and P-40 (p < 0.001) was significantly higher than at P-0. During the MRI, the SAP of all treatments was significantly lower during PROC than for other phases (p < 0.001), as well as lower than CT1 (p < 0.001) and CT2 (p = 0.036). The DAP followed similar trends to SAP.

The MAP of all treatments was significantly higher at S-34 than for the rest of the time points (p < 0.001). The MAP in SED had imaging and treatment differences as follows:

A-M resulted in higher MAP during MRI than CT1 (p = 0.045); P-M resulted in higher MAP during CT1 (p = 0.009) and CT2 (p = 0.012) than MRI; MAP was higher in A-M than P-M during CT1 (p = 0.049). There were no significant treatment differences for MAP during IND; however, the MAP at I-0 was significantly higher than I-5 to I-20 (p < 0.001) for CT2 and

MRI, and higher than I-10 to I-20 in CT1 (p < 0.001).

Differences in MAP were associated with the phase of the research but not the treatment. The MAP was significantly lower at the time of PROC than for other phases (p <

0.001). Despite the lack of a treatment effect, the MAP of P-M, A-S, and A-M increased significantly at END after the MRI, but remained low in the P-S treatment.

Comparisons between imaging modalities showed a higher MAP during both the CT1

(p = 0.013) and CT2 (p < 0.001) than during the MRI in A-S; a higher MAP during the CT1

124 than during the CT2 (p < 0.001) and MRI (p < 0.001) in A-M; and a higher MAP during CT1 than during the MRI (p < 0.001) in P-S. In addition, the MAP of A-M during the CT1 was higher than for P-M (p = 0.043); the MAP of A-S during the CT2 was higher than for A-M (p

= 0.012).

The CIL and CIP were similar between treatments (Table 7 and Table 8) and both were significantly higher during SED and IND than during END (p < 0.001).

The SVIL during SED was significantly higher than during IND (p < 0.001) and END

(p < 0.001). The SVRIL during SED and END was significantly higher than during IND (p <

0.001).

The need for dopamine was not significantly different between treatments and imaging modalities, despite the use of dopamine to treat hypotension in 30.4% of cases in the

MRI during PROC as follows: 10% in A-S; 0% in A-M, 22.2% in P-S, and 44.4% in P-M.

Dopamine was not necessary during the CT1 and CT2.

Respiratory variables are presented in Table 9. The fR was significantly higher in all treatments at S-34 than at the other time points (p < 0.001), and the fR at I-0 was significantly lower than at other time points (p < 0.001). There were no significant differences in the occurrence of apnea between treatments: A-S (9 out of 10 dogs), A-M (6 out of 9), P-S (5 out of 9), and P-M (7 out of 9). The PE’CO2 was not significantly different between treatments, but was higher during IND than PROC (p < 0.001) and END (p < 0.001), and PROC was significantly higher than END (p = 0.037).

There were no significant treatment differences in blood gas analysis, but there were significant differences between phases (Table 10). The pH was significantly higher during

SED than IND (p < 0.001) and END (p < 0.001), and significantly higher during END than

IND (p < 0.001). The PaCO2 was significantly higher during IND than SED (p < 0.001) and

END (p < 0.001), and significantly higher during END than SED (p < 0.001). The PaO2 was

125 significantly higher during END than SED (p < 0.001) and IND (p = 0.018), and significantly higher during IND than SED (p < 0.001). The Na+ was significantly higher during IND than

SED (p < 0.001) and END (p = 0.002). The K+ was significantly higher during END than

SED (p = 0.001) and IND (p < 0.001), and significantly higher during SED than IND (p <

0.001). The Cl- was significantly higher during SED (p < 0.001) and IND (p < 0.001) than

END. The Hb was significantly higher during SED (p < 0.001) and IND (p < 0.001) than

END. The Hb of A-S was significantly higher than P-M (p = 0.024). The lactate was significantly higher during IND than SED (p < 0.001) and END (p < 0.001). The temperature was significantly higher during SED than END (p < 0.001) and IND (p < 0.001), and was significantly higher during IND than END (p < 0.001).

126

DISCUSSION

This investigation demonstrated no differences in cardiovascular or respiratory variables during induction with propofol or alfaxalone with or without midazolam co-induction in healthy fentanyl sedated research dogs. In addition, no difference in cardiovascular variables was noted with either propofol or alfaxalone TIVA with mechanical ventilation during diagnostic imaging or in the recovery phase. Despite a dose reduction in requirements for propofol and alfaxalone demonstrated with midazolam co-induction

(Hopkins et al. 2013; Robinson & Borer-Weir 2013; Sanchez et al. 2013; Liao et al. 2015a) we did not demonstrate a benefit in cardio-pulmonary function. Currently only a few investigations have assessed cardio-pulmonary variables during induction. One study compared the effects of propofol with or without midazolam co-induction on indirect blood pressure in healthy dogs and demonstrated lower blood pressures when midazolam co-induction was included (Hopkins et al. 2013). We did not demonstrate a difference in blood pressure using a more accurate method of direct measurement. Moreover, additional measurements, not previously reported and included in our study, of cardiac output and derived hemodynamic variables were also similar between treatments receiving co-induction midazolam and those receiving the induction anesthetic alfaxalone or propofol alone.

One potential explanation for the lack of significant improvement in hemodynamic function may be related to the use of healthy research dogs, since the decrease in cardio-pulmonary function after induction with potent anesthetics mainly originates from sympatholysis, rather than direct injectable anesthetic depression. It has been demonstrated that within a clinical range of propofol plasma concentrations (less than 10 µg mL-1) (Joubert

2009), direct myocardial depression and arterial vasodilation is minimal (Ismail et al. 1992;

Nakamura et al. 1992; Belo et al. 1994) in dogs. An earlier study also demonstrated that the

127 decrease in systemic vascular resistance caused by propofol was more pronounced after autonomic abolishment than within clinical plasma concentrations in dogs (Goodchild &

Serrao 1989). The decrease in HR, MAP, and CI observed immediately after induction (I-0) may also be related to physiological changes, since similar drops of 10% – 20% in HR, MAP, and CI have been determined in chronically instrumented healthy research dogs after falling asleep and undergoing sympatholysis (Schneider et al. 1997).

There are no similar reports detailing the cardiovascular effects of alfaxalone during induction. However, in a study comparing cardio-pulmonary function after induction with tiletamin/zolazepam, ketamine/diazepam, propofol, and alfaxalone in healthy research dogs, no differences were noted (De Caro Carella et al. 2015). It is important to note that healthy research dogs might not be a suitable model to represent the cardio-pulmonary effects of induction anesthetics in sick animals, and that the possible benefits of co-induction agents may only be apparent in sick and critically ill patients.

Arterial blood pressure is a useful and commonly used variable to assess the safety of anesthesia. In our study, MAP gradually decreased to the 60 – 70 mmHg range after induction, and remained in that range during the imaging procedures, similar to other studies comparing propofol and alfaxalone in healthy research dogs without surgery (Ambros et al.

2008). Higher MAP value of 70 – 110 mmHg have been reported in another study comparing propofol and alfaxalone TIVA in client-owned dogs undergoing ovariohysterectomy (Suarez et al. 2012). Sympathetic stimulation from surgery is responsible for this difference and also demands a higher TIVA rate (Ambros et al. 2008; Suarez et al. 2012; Reed et al. 2015).

Considering these differing study results, the impact of stimulation needs to be taken into consideration when interpreting the cardiovascular data in anticipation of what would be noted in clinical patients.

Midazolam co-induction decreased the TIVA rate of propofol throughout the

128 maintenance period of anesthesia, but this reduction in dose was not associated with cardiovascular differences between P-S and P-M in our study. Similar findings have been shown in human patients in which MAP was even lower if a midazolam infusion was added to the maintenance rate in addition to its use as co-induction (Adams et al. 2002).

Interestingly, the MAP during MRI tended to be lower than during CT in all treatments except for the P-M treatment. In addition, MAP was lower during the MRI imaging than during any other study phase in all treatments. In a similar study using patients with clinical neurological disease, there were no differences in MAP between the MRI imaging and other phases of anesthesia, and MAP remained within 80 – 90 mmHg in that study, although some patients required dopamine support (Caines et al. 2014). Compared to the current study, in which MAP was approximately 60 during MRI, the propofol rate used was similar and the same criteria were used to institute a dopamine infusion to treat for hypotension. Therefore it seems likely that differences in MAP between the two studies are related to several factors, including the health status of the animal and sympathetic drive, which is likely increased in critical or compromised patients. Another possible explanation for the lower MAP during the MRI with respect to the CT scans is the noise generated by the

MRI, which may affect the depth of anesthesia and the dose of TIVA for maintenance. In human patients under propofol sedation, noise has been shown to increase consciousness

(Kim et al. 2001) and less propofol is required when the noise is blunted (Tharahirunchot et al. 2011). We placed earplugs in our dogs during the MRI scan and a lower rate was required during the imaging for the MRI than the CT1, but not the CT2. Additionally, although CT image acquisition is quieter then MRI, CT image acquisition involves movement of the patient table within the gantry, which can reasonably be expected to be stimulating. Another factor contributing to differences in MAP is the dorsal positioning of dogs in MRI versus sternal for CT. Cardio-pulmonary effects between right lateral and dorsal recumbency

129 showed that HR was 33% higher, SAP, MAP and DAP were 30% lower, and systemic vascular resistance was 17% lower in dogs in dorsal recumbency (Gartner et al. 1996).

A decrease in CI associated with a decrease in HR was observed after completion of the procedures in all treatments. Dogs were positioned in lateral recumbency for recovery and the decrease in HR could be the result of an arterial baroreflex that resulted from an increase in SVRI secondary to increased sympathetic activity from the stimulation of being moved.

There were no significant differences regarding the occurrence of apnea or hypoventilation between all treatments, similar to other studies (Ambros et al. 2008;

Amengual et al. 2013; Maney et al. 2013). Apnea with alfaxalone does not occur in healthy unsedated dogs until 10 times the clinical dose is administered (20 mg kg-1), while propofol caused apnea in 2 out of 6 dogs with 5 times clinical dose (32 mg kg-1) (Keates & Whittem

2012). The occurrence of apnea and hypoventilation are dose-dependent in both propofol and alfaxalone (Muir & Gadawski 1998; Muir et al. 2008; Keates & Whittem 2012) and midazolam co-induction did reduce both propofol and alfaxalone induction dose and P TIVA rate. However, midazolam causes respiratory depression in the presence of other anesthetics

(Heniff et al. 1997) even though it is clinically deemed to cause minimal respiratory depression when used alone.

In conclusion, this study demonstrates that, despite a reduction in the induction and

TIVA dose of propofol or alfaxalone when midazolam is used as a co-induction drug, there was no significant difference between treatments in cardio-pulmonary variables in healthy research dogs during the induction phase, TIVA maintenance for advanced imaging, or the recovery phase. The MAP pressure during MRI with dorsal positioning was lower than during CT imaging, necessitating dopamine infusion in 30% of cases. Overall, the cardiovascular effects of TIVA with propofol and alfaxalone for imaging were comparable in the current study.

130

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Figure 2. Study timeline with administration of fentanyl (F); propofol (P) or alfaxalone (A) with either saline (S) or midazolam (M); initiation (//) and discontinuation (//) of total intravenous anesthesia (TIVA) indicated. The arrows between phases indicated transfer of dogs and hence interruption of monitoring.

135

Figure 3. Median and 95% confidence interval for (a) heart rate (HR), (b) mean (MAP) arterial pressure in dogs randomly assigned to one of the four treatments: Propofol (P) +

Midazolam (M) (PM); Alfaxalone (A) + M: (AM); P + saline (S) (PS); and A + S (AS).

Treatments were as follows: Fentanyl (F) (7 µg kg-1, IV) was administered 10 minutes prior to an initial IV bolus of P (1 mg kg-1) or A (0.5 mg kg-1). Either M (0.3 mg kg-1, IV) or S was administered immediately after the initial P or A bolus followed by additional boluses of the respective induction drug every 6 seconds to allow endo-tracheal intubation and then followed by total intravenous anesthesia with the same induction anesthetic. Time points are presented with phase-time, in which phases include SED, 30 minutes before sedation with F

(S-30) and 4 minutes after F (S-34); IND, at the time of intubation after administration of the induction anesthetic (I-0), and 5 (I-5), 10 (I-10), and 15 (I-15) minutes while breathing spontaneously, and at 20 (I-20) minutes once on mechanical ventilation; PROC, at the beginning of the imaging procedure (P-0) and every 5 minutes for up to 40 minutes (P-40) during imaging; and END, once the dog returned to the induction area (E-0) and 5 minutes later (E-5), prior to recovery . The dotted vertical line indicates induction.

136

Propofol + Saline (a) Propofol + Midazolam

175 Alfaxalone + Saline Alfaxalone + Midazolam

150

) 125 -1

100

75 HR (beats minute

S-30 S-34 I-0 I-5 I-10 I-15 I-20 P-0 P-5 P-10 P-15 P-20 P-25 P-30 P-35 P-40 E-0 E-5 Phase-Time (minutes)

(b) 150 Propofol + Saline Propofol + Midazolam

125 Alfaxalone + Saline Alfaxalone + Midazolam

100

75 MAP (mmHg) MAP

0 S-30 S-34 I-0 I-5 I-10 I-15 I-20 P-0 P-5 P-10 P-15 P-20 P-25 P-30 P-35 P-40 E-0 E-5 Phase-Time (minutes)

137

Table 6. Median and 95% confidence interval for systolic (SAP) and diastolic (DAP) blood pressure. (see Figure 3 for key).

PS*+ PM+ AS* AM+ 142.5 139.7 149.3 140.3 S-30a (128.8 - (126.4 - 154.4) (134.3 - 165.9) (127.6 - 154.3) 157.7) 170.6 171.6 167.5 161.5 S-34A (154.2 - (155.3 - 189.6) (150.7 - 186.2) (146.2 - 178.3) 188.8) 120.1 128.5 126.4 134.4 I-0aB (108.7 - (116.5 - 141.6) (113.4 - 141) (122.3 - 147.6) 132.7) 112.8 119.7 112.7 121.7 I-5ab (102.1 - (108.6 - 132) (101.7 - 125) (110.9 - 133.7) 124.5) 109.9 103.1 114.7 105.4 I-10ab (99.7 - 121.2) (93 - 114.3) (104.5 - 125.9) (95.5 - 116.4) 100.3 97.1 109.8 101.5 I-15abC (91 - 110.6) (87.6 - 107.6) (100 - 120.6) (91.9 - 112.1) 93.6 86.6 108.7 87.0 I-20abc (84.9 - 103.2) (78.0 – 96.0) (99 - 119.4) (78.7 - 96.1) 92 85.2 91.3 81.4 P-0aD SAP (83.6 - 101.3) (76.8 - 94.4) (83.2 - 100.3) (73.7 – 90.0) (mmHg) 85.7 80.2 91.9 83.9 P-5a (77.8 - 94.4) (72.4 - 88.9) (83.7 - 100.8) (75.9 - 92.7) 84.4 81.2 92.1 83.8 P-10a (76.7 – 93.0) (73.4 – 90.0) (83.9 - 101.1) (75.9 - 92.6) 85.5 81.6 98.4 82.9 P-15a (77.6 - 94.2) (73.7 - 90.4) (89.7 - 108) (75.1 - 91.5) 89.5 81.3 97.9 84.9 P-20a (81.2 - 98.5) (73.4 – 90.0) (89.2 - 107.4) (77.0 - 93.7) 92.3 84.7 95.4 87.4 P-25a (83.8 - 101.6) (76.5 - 93.8) (87.0 - 104.7) (79.2 - 96.4) 97.4 85.8 99.8 92.3 P-30a (88.5 - 107.3) (77.5 – 95.0) (91.0 - 109.5) (83.6 - 101.8) 104.9 94.2 104 95.3 P-35ad (95.2 - 115.5) (85.1 - 104.4) (94.8 - 114.1) (86.4 - 105.2) 104.6 102.9 106.6 98.6 P-40ad (94.5 - 115.6) (92.9 - 114.1) (96.7 - 117.5) (89.3 - 108.9) 112.5 100.1 114.8 102.5 E-0a (101.9 - 124.2) (90.1 - 111.2) (104.5 - 126.2) (92.7 - 113.4) 99.9 99.1 110.3 97.4 E-5a (90.5 - 110.2) (89.2 - 110.1) (100.4 - 121.2) (88.1 - 107.8) 87.7 91.9 85.6 87.6 S-30a DAP (79.5 - 96.7) (82.8 - 102.1) (78.0 – 94.0) (79.3 - 96.8) (mmHg) 91.2 92.1 85.2 87.8 S-34A (82.7 - 100.6) (83.0 - 102.3) (77.3 - 93.8) (79.5 – 97.0) 138

61.1 71.2 69.2 64.6 I-0aB (55.5 - 67.2) (64.0 - 79.2) (63.1 - 75.9) (58.5 - 71.3) 60.2 62.3 59.8 60.5 I-5ab (54.7 - 66.3) (56.3 – 69.0) (54.5 - 65.6) (54.9 - 66.7) 55.5 57.5 57.9 56.0 I-10ab (50.4 - 61.1) (51.9 - 63.6) (52.9 - 63.5) (50.8 - 61.7) 52.9 52.7 56.1 54.7 I-15ab (48.0 - 58.2) (47.6 - 58.4) (51.2 - 61.5) (49.6 - 60.3) 50.8 50.5 55.7 51.3 I-20ab (46.1 - 55.9) (45.6 – 56.0) (50.8 - 61.1) (46.5 - 56.6) 53.6 50.1 54.6 54.7 P-0aC (48.7 - 58.9) (45.3 - 55.5) (49.8 - 59.8) (49.6 - 60.3) 53.2 52.2 55.5 55.4 P-5a (48.4 - 58.5) (47.2 - 57.7) (50.7 - 60.8) (50.2 - 61.1) 53.6 53.7 57.3 54.3 P-10a (48.7 - 58.9) (48.6 - 59.4) (52.3 - 62.7) (49.3 - 59.9) 54.8 53.9 59.4 54.7 P-15a (49.9 - 60.3) (48.8 - 59.6) (54.2 – 65.0) (49.7 - 60.3) 57.3 52.8 59.4 54.2 P-20a (52.1 – 63.0) (47.7 - 58.4) (54.2 - 65.1) (49.2 - 59.7) 57.7 54.5 58.2 55.9 P-25a (52.5 - 63.4) (49.3 - 60.2) (53.1 - 63.7) (50.8 - 61.6) 58.4 54.0 60.6 59.3 P-30ac (53.1 - 64.2) (48.8 - 59.7) (55.3 - 66.4) (53.8 - 65.3) 60.3 60.7 62.1 60.3 P-35ac (54.9 - 66.3) (54.9 - 67.1) (56.7 – 68.0) (54.7 - 66.4) 60.8 66.7 61.8 59.5 P-40ac (55.1 - 67.1) (60.2 - 73.8) (56.2 – 68.0) (54.0 - 65.6) 57.5 60.5 61.9 60.3 E-0aD (52.1 - 63.3) (54.6 - 67.1) (56.4 - 67.9) (54.6 - 66.6) 51.9 55.3 58.0 55.9 E-5ad (47.1 - 57.2) (49.8 - 61.3) (52.9 - 63.7) (50.7 - 61.8)

Same superscript letters but different case indicates there is a significant difference between time points. Different superscript symbols (* and +) indicates significant difference between treatments of SAP (p < 0.05).

139

Table 7. Median and 95% confidence interval for cardiac index (CIL), stroke volume index

(SVIL) and systemic vascular resistance index (SVRIL) calculated from cardiac output (CO) measured by LiDCO. The CO was measured 4 minutes after sedation with fentanyl (7 µg kg-1,

IV) prior to induction (SED), 15 minutes after induction (IND) and 5 minutes after returning to the induction area prior to recovery (END). (see Figure 3 for key).

PS PM AS AM SED 158.50 123.44 136.29 172.92 CIL (124.68 - 201.50) (98.27 - 155.05) (111.22 - 167.02) (140.54 - 212.77) (mL -1 IND 133.45 117.25 122.16 136.44 kg -1 (107.99 - 164.91) (96.05 - 143.14) (100.65 - 148.25) (110.88 - 167.88) min ) END* 80.07 77.64 86.94 81.71 (64.8 - 98.95) (63.6 - 94.78) (71.63 - 105.51) (66.41 - 100.54) SED* 1.59 1.51 1.56 1.76 SVIL (1.32 - 1.93) (1.27 - 1.79) (1.34 - 1.82) (1.50 - 2.06) (mL kg-1 IND 1.46 1.27 1.29 1.10 beat-1) (1.24 - 1.73) (1.09 - 1.48) (1.12 - 1.50) (0.94 - 1.29) END 1.30 1.29 1.34 1.17 (1.10 - 1.54) (1.11 - 1.50) (1.12 - 1.55) (0.99 - 1.37) SED 0.67 0.84 0.75 0.63 SVRIL (0.53 - 0.86) (0.66 - 1.06) (0.61 - 0.93) (0.51 - 0.78) (mmHg mL-1 IND* 0.40 0.52 0.52 0.47 min-1 (0.35 - 0.54) (0.42 - 0.64) (0.43 - 0.64) (0.38 - 0.58) kg-1) END 0.75 0.79 0.75 0.75 (0.60 - 0.93) (0.64 - 0.97) (0.62 - 0.92) (0.60 - 0.92)

There was a significant difference between phases but not between treatments.

* Significantly different from the other two phases (p < 0.05).

140

Table 8. Median and 95% confidence interval for cardiac index (CIP) calculated from cardiac output (CO) measured by PulseCO. Measurement was discontinued during imaging procedure. (see Figure 3 for key)

PS PM AS AM 167.6 140.8 138.4 132.1 S-34ab (126.5 - 222) (102.1 - 194.2) (103.6 - 184.8) (100.1 - 174.2) 185 136.8 151.8 178.9 I-0ab (143.5 - 238.7) (103.1 - 181.4) (113.5 - 203) (137.6 - 232.6) 155.4 129.2 158.6 162 I-5ab (120.5 - 200.5) (97.2 - 171.7) (119 - 211.5) (124.8 - 210.4) 187.4 154.6 171.9 197 CIP I-10ab (145.3 - 241.7) (117 - 204.2) (129 - 229.2) (151.7 - 255.8) (mL kg-1 170.3 149.4 132.6 145.5 min-1) I-15ab (132.1 - 219.7) (113.1 - 197.4) (99.4 - 176.8) (112.1 - 189) 160.96 129.72 133.14 119.81 I-20ab (124.8 - 207.62) (97.84 - 172) (99.55 - 178.07) (92.14 - 155.78) 102.5 58.8 65.9 58.8 E-0a (79.5 - 132.2) (44.3 - 78) (50.2 - 86.5) (45.2 - 76.4) 86.5 69.2 63.6 63 E-5b (67.1 - 111.6) (52.3 - 91.4) (48.6 - 83.3) (48.5 - 81.9)

Different superscript letters indicates there is a significant difference between time points. (p

< 0.05).

141

Table 9. Mean and 95% confidence intervals (CI) for respiratory rate (fR) and median and

95% CI end-tidal carbon dioxide (PE’CO2). The fR from I-0 – I-15 corresponds to spontaneous ventilation. The PE’CO2 during Proc and End are presented as the summation of the whole phase. (see Figure 3 and Table 6 and Table 7for key)

PS PM AS AM fR S-0a 24.2 29.3 27.8 31.6 (min-1) (19.1 – 29.2) (24.9 – 33.7) (23.5 – 32.0) (27.6 – 35.7) S-30a 26.6 29.3 22.9 28.1 (22.6 – 30.5) (24.9 – 33.7) (18.8 – 27.0) (24.1 – 32.1) S-34a 27.5 30.5 30.0 31.5 (20.7 – 34.3) (23.8 – 37.2) (25.3 – 34.7) (25.9 – 37.1) I-0b 6.9 2.0 0.5 6.1 (3.2 – 10.6) (-2.2 – 6.2) (-0.3 – 4.2) (2.3 – 9.9) I-5c 14.8 9.2 6.0 7.0 (11.1 – 18.5) (5.0 – 13.4) (2.2 – 9.7) (3.1 – 10.8) I-10c 12.4 9.0 9.4 4.5 (8.6 – 16.2) (4.8 – 13.2) (5.6 – 13.1) (0.7 – 8.3) I-15c 9.2 7.1 9.5 7.0 (5.5 – 12.9) (2.9 – 11.3) (5.7 – 13.2) (3.2 – 10.9) a PE’CO2 I-0 33.9 40.3 39.0 38.6 (mmHg) (30.6 – 37.1) (36.5 – 44.1) (35.2 – 42.9) (34.7 – 42.5) I-5b 38.8 40.1 42.3 46.3 (35.5 – 42.0) (36.4 – 43.9) (39.0 – 45.6) (42.4 – 50.2) I-10c 43.7 45.2 47.0 48.9 (40.3 – 47.1) (41.6 – 48.7) (43.8 – 50.3) (45.2 – 52.7) I-15d 47.5 50.3 48.8 52.9 (44.1 – 50.9) (46.7 – 53.8) (45.5 – 52.1) (49.4 – 56.4) Proc 39.1 39.8 40.8 39.3 (37.4 – 40.9) (37.8 – 41.7) (39.2 – 42.4) (37.6 – 41.0) End 37.9 38.6 39.2 38.6 (35.5 – 40.3) (36.0 – 41.1) (37.0 – 41.3) (36.2 – 40.9)

Different superscript letters indicates there is significant difference between time points (p <

0.05).

142

Table 10. Median and 95% confidence interval for temperature and arterial blood gas analysis, including pH, arterial carbon dioxide (PaCO2), oxygen (PaO2) partial pressure, sodium (Na+), potassium (K+), chloride (Cl-), hemoglobin (Hb), and lactate (Lac) concentration. (see Figure 3 and Table 6 for key).

PS PM AS AM pH SEDa 7.37 7.36 7.36 7.35 (7.35 - 7.40) (7.34 - 7.39) (7.34 - 7.39) (7.33 - 7.38) INDb 7.22 7.21 7.24 7.21 (7.20 - 7.25) (7.18 - 7.24) (7.22 - 7.27 (7.18 - 7.23) ENDc 7.31 7.30 7.31 7.31 (7.28 - 7.34) (7.28 - 7.33) (7.2 9 - 7.34) (7.28 - 7.34) a PaCO2 SED 35.0 37.2 37.0 37.7 (mmHg) (31.3 - 38.7) (33.6 - 40.7) (33.5 - 40.5) (34.2 - 41.3) INDb 53.9 55.9 50.0 56.2 (50.2 - 57.6) (52.2 - 59.7) (46.6 - 53.4) (52.7 - 59.8) ENDc 43.0 43.6 43.3 43.0 (39.4 - 46.7) (39.8 - 47.3) (39.8 - 46.8) (39.3 - 46.7) a PaO2 SED 86.1 83.8 84.3 86.6 (mmHg) (76.8 - 96.5) (75.3 - 93.2) (75.8 - 94.0) (77.8 - 96.3) INDb 517.2 503.2 486.8 457.5 (461.5 - 579.6) (449.6 - 563.3) (439.5 - 539.1) (411.0 - 509.2) ENDc 544.3 520.8 542.1 546.8 (485.7 - 610.0) (465.3 - 582.9) (486.8 - 603.6) (488.2 - 612.4) Na+ SEDa 146.8 146.4 145.7 145.4 (mmol (145.5 - 148.0) (145.0 - 147.7) (144.5 - 146.9) (144.1 - 146.7) L-1) INDb 147.9 147.3 147.0 147.6 (146.7 - 149.1) (146.0 - 148.6) (145.8 - 148.2) (146.3 - 148.8) ENDa 146.0 147.1 146.7 146.9 (144.8 - 147.2) (145.7 - 148.4) (145.4 - 147.9) (145.6 - 148.1) K+ SEDa 3.7 3.7 3.6 3.8 (mmol (3.5 - 3.9) (3.5 - 3.8) (3.5 - 3.8) (3.6 - 3.9) L-1) INDb 3.4 3.3 3.3 3.4 (3.2 - 3.6) (3.1 - 3.5) (3.1 - 3.4) (3.2 - 3.6) ENDc 4.0 3.9 3.7 3.8 (3.8 - 4.1) (3.7 - 4.1) (3.5 - 3.8) (3.7 - 4.0) Cl- SEDa 118.3 117.0 117.4 117.5 (mmol (117.3 - 119.3) (115.9 - 118.0) (116.4 - 118.4) (116.5 - 118.5) L-1) INDa 118.1 117.1 117.4 117.3 (117.1 - 119.1) (116.1 - 118.2) (116.4 - 118.4) (116.3 - 118.3) ENDb 116.2 115.5 116.2 116.2 (115.2 - 117.2) (114.4 - 116.6) (115.2 - 117.2) (115.2 - 117.3) Hb SEDa 16.2 16.6 16.2 16.5 (g dL-1) (15.3 - 17.3) (15.6 - 17.6) (15.2 - 17.1) (15.6 - 17.5)

143

INDa 15.8 16.8 15.5 16.0 (14.8 - 16.7) (15.8 - 17.9) (14.6 - 16.4) (15.1 - 17.0) ENDb 13.5 13.7 13.3 13.3 (12.7 - 14.3) (12.8 - 14.5) (12.6 - 14.2) (12.5 - 14.1) Lac SEDa 1.1 1.4 1.0 1.0 (mmol (0.7 - 1.5) (1.0 - 1.8) (0.6 - 1.3) (0.6 - 1.4) L-1) INDb 1.4 2.1 1.3 1.5 (1.0 - 1.8) (1.7 - 2.5) (1.0 - 1.7) (1.1 - 1.9) ENDa 0.8 0.9 0.8 1.0 (0.4 - 1.2) (0.5 - 1.3) (0.4 - 1.2) (0.6 - 1.4) Temp SEDa 38.6 38.8 38.5 38.7 (°C) (38.2 – 38.9) (38.4 – (38.2 – 38.9) (38.4 – 39.0) 39.1) INDb 37.5 37.8 37.4 37.7 (37.2 – 37.8) (37.4 – 38.1) (37.1 – 37.8) (37.4 – 38.0) ENDc 36.0 36.3 36.0 36.2 (35.7 – 36.3) (36.0 – 36.7) (35.7 – 36.3) (35.9 – 36.5)

Different superscript letters indicate there is a significant difference between phases (p <

0.05).

144

CHAPTER IV

GENERAL DISCUSSION AND CONCLUSIONS

Research to assess the combined effects of primary induction agents with co-induction agents is warranted to fully define the interactions between the drugs in terms of dose requirements of the induction anesthetic and its cardio-pulmonary effects. The premise is that reducing the dose of the induction anesthetic can potentially reduce the depressive cardio-pulmonary effects and their duration. However, the inherent cardio-pulmonary effects of co-induction agents need to be considered and may result in additive or synergistic effects with those of the induction anesthetic.

This research was initiated to answer if midazolam, used as a co-induction agent, could reduce the dose of two current primary induction anesthetics, propofol and alfaxalone.

If a reduction in dose was achieved, an additional aim was to determine if it would also ameliorate any the potential negative cardio-pulmonary effects of these induction anesthetics.

In our study, the use of midazolam co-induction in fentanyl sedated healthy research dogs improved the induction quality and reduced the induction dose requirement of propofol and alfaxalone. In addition, for propofol but not alfaxalone, midazolam co-induction also reduced the maintenance dose required for total intravenous anesthesia (TIVA) to perform advanced imaging for up to one hour. Despite dose reductions in propofol or alfaxalone during induction and/or maintenance, there were no cardio-pulmonary differences for propofol or alfaxalone treatments between those that did receive midazolam and those that did not, and no significant recovery quality differences. This research has therefore answered the question that, in healthy dogs, the dose reduction in propofol and alfaxalone caused by midazolam co-induction does not result in any benefit in cardio-pulmonary function.

The effect of a higher dose of midazolam on the dose of propofol or alfaxalone was not assessed in this study. We tested midazolam at 0.3 mg/kg, IV, administered after the

145 initial bolus of the induction anesthetic. It has been shown that midazolam co-induction with propofol is more effective when midazolam is given at doses of 0.2-0.5 mg/kg, after an initial bolus of propofol (Robinson & Borer-Weir 2013; Sanchez et al. 2013). Similar information for alfaxalone was not available before our study, except for one observational study (Seo et al. 2015). This study is the first to demonstrate that midazolam co-induction with alfaxalone reduces the induction dose in healthy dogs.

In our study we measured blood pressure and cardiac output as part of the cardiovascular assessment for the effects of injectable anesthetics and the influence of co-induction with midazolam. Blood pressure and cardiac output are not always correlated due the interplay between pressure, resistance, and flow. In this study, there was no significant difference for mean arterial pressure (MAP) between treatments. A trend, although not significant, was observed for saline treatments to have a higher MAP than treatments that received midazolam, as well as for both alfaxalone treatments to have higher

MAP than the propofol treatments during the imaging phase. Our prospective power analysis estimated that with regard to MAP, 8 dogs were required in each treatment to denote a 10 mmHg difference in the mean value for MAP and a standard deviation of 7, with a power of 80%, and an α level of 0.05. We used 9 to 10 dogs in each treatment; the difference in the MAP of all treatments was generally less than 10 mmHg and the MAP was always higher than 60 mmHg, which is considered an acceptable lower limit for MAP (Ruffato et al.

2015). Therefore, our conclusion is that regardless of the trends observed, all protocols resulted in clinically acceptable MAP.

Cardiac output (CO) values were similar between all treatments. Cardiac output is measured with less frequency than direct blood pressure in clinical cases; however in the research setting, CO provides important information that can reflect on tissue perfusion and requirements. In our study, CO decreased significantly at the end of the imaging procedure

146 before the recovery phase, associated with a decrease in heart rate and no change in stroke volume. This change was short lasting and did not impact tissue perfusion considering that lactate measurements did not change when compared to periods in which the CO was higher.

Normal values reported for CO and standardized as cardiac index (CI) in conscious dogs vary by as much as 25% (Gerard et al. 1990; Haskins et al. 2005), probably as a result of body function, including sympathetic tone, activity of the dog, and tissue demands, but also may depend on the method used to determine the CO. Cardiac output can drop by as much as

20% during sleep (Schneider et al. 1997), and with this in mind it should be considered that

CO is always adequate if it meets the demands of the tissues. Likewise, in an anesthetized patient, CO values decrease due to changes in sympathetic tone and vascular resistance, in addition to the effects of anesthetics on myocardial contractility. The lowest values measured prior to the recovery period ranged between 78-87 mL kg min-1, compared to higher values measured during the sedation phase (123-173 mL kg min-1) and maintenance phase (117-136 mL kg min-1). Despite these differences, values for CI in our study were considered clinically acceptable at all times.

In conclusion, based on measured MAP and LiDCO, all treatments had acceptable clinical values and therefore these is no evidence to favor any of the induction and TIVA protocols for this type of research population using ASA 1 patients.

LIMITATIONS OF THE STUDY

There are some potential limitations of this study. First, the use of an incomplete

Latin square crossover design that included three types of imaging procedure, namely MRI and CT with or without intervertebral disc injection, may decrease the power of this study because of patient allocation. The reason for this design was to incorporate the research with another parallel but unrelated study and meet the 3R principle: reduce, replace and

147 refine, according to the recommendations of animal use in research. Despite the use of appropriate statistical models used to analyze the data from this study design, which included a general linear mixed model, there were only 5 dogs in the CT2 and only one dog in AM, PS and PM treatment each, which lowers the power of those treatments for definitive conclusions.

Another limitation was the injection of the induction anesthetic drug by intermittent hand injection for the initial bolus and additional doses, instead of administration by constant rate infusion using a syringe pump. In theory, a constant rate infusion by pump provides better control of the rate and reduces human error and bias. However, this study intended to provide a clinical scenario and it was deemed important to mimic those conditions by performing the injections by hand.

During the maintenance phase, anesthesia depth assessment could only be done intermittently for the safety of personnel during the imaging. This could impact the dose requirements during that phase because the assessments had to be interrupted and close monitoring was not possible. In addition, the requirements determined in this study refer to diagnostic procedures, which are different from those that result from surgical stimulation.

Another consideration was how the monitoring equipment was adapted among the different imaging procedures. First, the length of extension tubing for direct arterial blood pressures differs significantly between MRI and CT procedures, and also during the MRI acquisition and other the other phases. Different lengths of extension tubing could result in different natural frequencies and damping coefficients within the pressure monitoring system, affecting the blood pressure waveform and readings (Miller 2015). All precautions and recommended materials were used during the measurement of cardiovascular parameters to reduce the impact of these technical differences. Second, we used different monitors for the different phases and imaging procedures due to availability of the monitors. It is expected

148 that minimum discrepancy occurs between the different monitors and calibrations were always performed prior to use.

Finally, there was a one-year gap between first and second (third MRI and second CT) round of data collection (see for CONSORT flow diagram). During this time, dog numbers changed due to the adoption of some of them. Also one of the dogs participated only in the second round of data collection. Due to the concern of further lowering the number of subjects in each treatment, the third MRI data were pooled with first and second MRI for analysis, while the second CT was analyzed separately since significant differences were expected from the injection performed in the unrelated study. This could have created some bias considering the TIVA rate and quality of anesthesia of CT2 was significantly lower than

CT1, even after induction before the imaging procedure commenced.

STRENGTHS OF THE STUDY

In this study, descriptive scoring systems were employed for a more objective evaluation of sedation, induction and intubation, maintenance, extubation, and recovery.

Compared to subjectively assigning a qualitative evaluation, a descriptive scoring system describes different aspects of the event and allows for better post hoc comparisons. Moreover, we included videotaping of each event with the descriptive scoring system with multiple blinded researchers doing the scoring, allowing post hoc comparisons and improving the validity of the evaluation by minimizing subjective bias.

To date, many studies have mask induced the animals with inhalant, instrumented them while anesthetized and later recovered the patients before initiating the data collection.

The research dogs in this study were acclimatized to the anesthesia preparation area and topical local anesthetic cream and lidocaine infiltration were used to desensitize the catheterization sites. This facilitated the instrumentation for direct arterial blood pressures

149 and cardiac output (LiDCO and PulseCO) measurements in conscious dogs. This process was not stressful to the dogs, was repeatable with multiple arterial catheters placed over the crossover design, and in the author’s opinion was less stressful to the animals compared to inhalant mask induction. Prior to data collection the dogs were sedated with fentanyl and the stress of a laboratory environment was further minimized prior to initiation of induction.

The size of the dog was also considered important for the repeatability of our methods, that is dogs > 15-20 kg are ideal. One beagle research dog, 7 kg, was originally enrolled in the study, however, due to her size, arterial catheterization was difficult while conscious and had to be completed after induction. Due to her size and loss of data, this dog was excluded from the study (see Figure 4for CONSORT flow diagram).

AREAS FOR FUTURE RESEARCH

This research was conducted in healthy research dogs and has provided substantial information as to how co-induction with midazolam impacts dose requirements for induction anesthetics and the resultant cardio-pulmonary effects in ASA I patients. However, the true goal is to assess the impact of co-induction agents in sick clinical cases, ASA 3 or higher.

Therefore, further investigations should include the following: different premedications; longer acting sedatives and/or more potent sedatives; different physiologic patient conditions

(such as hemorrhage, anemia, sepsis, and hypoxemia); different age populations (neonatal, pediatric, and geriatric); and different species (cats, rabbits, avian, ruminants, and equine).

Such studies would be more applicable to client-owned cases. In addition to the induction process and dosing, further investigations into the hemodynamic changes (decrease in HR and CO) during the final stages of advanced imaging in clinical cases warrants further research. Additional pharmacokinetic studies to support the pharmacodynamic observations of co-induction agents would also strengthen their use.

150

Future research projects with these questions in mind could include:

1. Investigation of midazolam co-induction in various subject species, populations,

conditions or premedications in the laboratory.

2. Investigation of midazolam co-induction in various subject species, populations,

conditions or with opioid/acepromazine premedication in clinical patients

3. Retrospective investigation of heart rate before, during and after diagnostic imaging

4. Investigation of the application of midazolam co-induction techniques with alfaxalone

and propofol by veterinary students, technicians, or general practitioners in cats or dogs.

5. Pharmacokinetic interactions of midazolam co-induction to propofol or alfaxalone

induction and TIVA doses.

151

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midazolam on the propofol dose requirements for induction of general anaesthesia in

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dogs? Results from a survey of diplomates of the ACVAA and ECVAA. Veterinary

Anaesthesia and Analgesia 42, 55-64.

Sanchez A, Belda E, Escobar M et al. (2013) Effects of altering the sequence of midazolam

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Schneider H, Schaub CD, Andreoni KA et al. (1997) Systemic and pulmonary hemodynamic

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152

Figure 4. Study CONSORT diagram demonstrates sample numbers in each imaging modalities including magnetic resonance imaging (MRI); computer tomography with intervertebral disc injection (CT1); computer tomography without intervertebral disc injection (CT2) and the treatments distribution: propofol (P) - saline (S); P – midazolam (M); alfaxalone (A) – S; AM.

153

Appendix 1: program codes for the general linear mixed model title &trans is the response and transform, if any; libname sasuser 'c:\sasdata'; data cardio; set sasuser.pentingtest; format device $4.; if group=11 then trt='AS'; if group=12 then trt='am'; if group=21 then trt='PS'; if group=22 then trt='pm' if tx in (1,3,5) then device = 'MRI'; if tx = 2 then device ='CT1'; if tx = 4 then device ='ct2'; if zone in ('pre','novent') then device='none'; if zone='endpro' and time=3 then delete; if zone='reco' and time=0 then delete; if zone='Vent' and time=45 or time=50 or time=55 then delete; if zone='pre' and time=33 then delete; if device='none' then delete proc sort; by sex dog trt zone time device; data pre33; set cardio; if zone='pre' and time=33; keep sex dog trt sec_scr saliva nausea defec device; data pre35; set cardio; if zone='pre' and time=35; keep sex dog trt ind_scr no_top ind_dose tpm_ind device; data cardio; set cardio; drop ind_scr no_top ind_dose tpm_ind sec_scr saliva nausea defec; data cardio; merge cardio pre33 pre35; by sex dog trt; format junk1 $6. junk2 $2.; drop junk1 junk2; junk1=zone; junk2=time; ZT=junk1||junk2; proc print;var dog zone time trt zt sex device lidco bwt cil lcil; proc mixed noitprint noclprint covtest cl method=reml; class dog zone time trt sex zt device; model cil = trt sex device /outp=new; *** starter model--start with this for each new response; *random zone(dog trt sex device); *** this statement or next but NOT both; * repeated / subject=zone(dog trt sex ) type=ar(1); *** types: ar(1) arh(1) sp(pow)(time) toep toep(2)-toep(11) toeph toeph(2)-toeph(11) un un(2)-un(11); lsmeans trt sex device / cl adjust=tukey tdiff; make lsmeans out=mean; data mean; set mean; drop stderr df alpha probt tvalue lower upper; LL=exp(lower); Median=exp(estimate); UL=exp(upper); proc print noobs; data diff; set diff; drop stderr df alpha tvalue lower upper; LL=exp(lower); Ratio=exp(estimate);UL=exp(upper); proc print noobs; proc univariate plot normal data=new; var resid; proc plot data=new; plot resid*(pred trt zone sex dog zt time device); run; quit;

154

Appendix 2.1: Raw data of dog 1

HR RR TEMP Attitude Date:______Oct 6___2014______Pre-Instrumentation 100 pant 38.5 nervous Randomization Dog number: ______Time Response Attempts Dog ID#: ______EMLA application Dog Name: ______Alonzo______Cephalic vein Meloxicam 1125 Dog Weight:______27______KG Dorsal pedal artery After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______Blood Gas:______126 42 173 106 84 4.7/4.8/4.9 Fentanyl Admin Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______1252______3.9 Yes No No 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 1259 89 108 185 104 80 3.1/3

BloodGas:______103 pant 191 106 77 3.71/CI 4.11 3.6/3.7/3.8 148 Time Starting Induction: ______1305______P/A Vol: ______Time: Time: Time: Time: Time: Time: Time Intubation: ______1307______1305 1306 1306 1306 1307 Mid/Sal Vol :______1 2 3 4 5 6 Total dose Intubation score: Injectable Volume ______Attempt to Intubate x x Comments Relaxed jaw tone Lateral palpebral Medial palpebral Coughing X X Swallowing X x Paddling Vocalization

155

POST- Out Alonzo 5 10 IPPV INTO 5 10 15 20 25 30 35 40 45 50 55 Pre-Recov TIVA 5 min 15 min of Oct/06/14 min min Starts MRI min min min min min min min min min min min ery OFF post Intub MRI

TIME 1307 1312 1317 1322 1327 1336 1341 1346 1351 1356 1401 1406 1411 1416 1421 1426 1431 1436 1443 1446 1457 Fluid Rate 135

250/70- 350/ 375/ 425/1 400/1 425/1 400/ P or A rate >325/91 98 105 19 12 19 112 P/A Top 1309,13 1312 1319 1336 1436 Up# 10,1311 HR bpm 75 75 105 90 112 100 97 174 176 177 163 187 104 108 99 101 96 103 80 85 92 83 110 SAP mmHg 129 122 107 98 93 97 81 73 60 71 75 91 101 107 111 125 129 130 129 101 94 94 118 DAP mmHg 51 63 55 51 49 50 58 56 50 58 59 67 73 74 79 86 89 94 58 50 50 49 64 MAP mmHg 71 74 68 64 61 65 66 64 54 65 66 77 82 86 89 100 104 106 77 63 62 62 78 o Temp C 38.2

SPO2 97 97 97 97 96 98 98 97 98 98 98 98 98 98 98 98 98 98 97 96 93

ET CO2 35 49 53 50 45 45 45 42 42 43 45 44 43 45 47 40 40 42 42 42 0->1 RR 0 19 11 10 12 12 12 12 12 12 12 12 12 12 12 12 10 10 10 5 2.19/CI LiDCO 2.42 3.5 2.1 3.2/3 3.1 2.6/2 1.8/1 2.4/2.6/ 2.3/2 /3. 3.1/3. 2.6/2 /2. 2.5/2.8/ PulseCO .3/3. /3. .7/2. .9/2/ 2.9 .4 6/3 2 .7 2/2 2.7 4 2 9 2.1 .7 .4 2.1/2 2.2/2. PulsCO 2.1/2.4 .4/2. 2.4/2. 4/2.7 IND 1min 5 5 3min 4min 2min Anesth 2 1 1 0 0 1 1 0 0 0 0 0 0 0 0 0 0 1 0

156

Alonzo Extub Stop TIVA E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/06/14 Time 0 Time 1446 1452 1457 1502 1507 1522 1537 1552 Score R0 R0 R0 P3 P3 P1 P0 Sternal: Other Stand:1537 1536

PlasmaSample

Alonzo 3min after Pre-Vent End MRI Oct/06/14 fentanyl Na+ 148 149 148 Cl - 122 121 118 K+ 3.5 3.6 4.3 Hb 19.1 16.5 14.8 pH 7.385 7.211 7.259 PCO2 31.2 55.8 49.5 PaO2 85.5 532 512 HCO3- 17.7 21.2 21.5 ABE -4.6 -6.9 -5.7 Lactate 2.1 1.4 0.9 PCV (%) 55 48 48 TP (mg/dL) 64 60 60 Plasma Sample:

157

HR RR TEMP Attitude Date:______Oct 14 2014______Pre-Instrumentation 100 20 38.4 BAR/nervous Randomization Dog number: ______Time Response Attempts Dog ID#: ______EMLA application 0930 Dog Name: ______Alonzo______Cephalic vein Good Meloxicam 1035 Dog Weight:______27.5______KG Dorsal pedal artery Left easy After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______Blood Gas:______107 12 164 118 96 3 Fentanyl Admin Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______1102______3.9 Yes No No 2 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 104 pant 217 123 93 3.1 BloodGas:______85 195 126 96 3 3.6/3.7/4/4. 79 187 116 88 3.73/CI 3.95 1/3.9/3.8 Time Starting Induction: ______1110______P/A Vol: ______Time: Time: Time: Time: Time: Time: Time Intubation: ______1111______1110 1111 1111 Mid/Sal Vol: ______1 2 3 4 5 6 Total dose Intubation score: Injectable Volume ______1______Attempt to Intubate x Comments Relaxed jaw tone Lateral palpebral Medial palpebral Coughing Swallowing Paddling Vocalization

158

POST- Out Alonzo 5 10 15 IPPV INTO 5 10 15 20 25 30 35 40 Pre-Recove TIVA 5 min post of Oct/14/14 min min min Starts MRI min min min min min min min min ry OFF extubation Intub MRI

123 TIME 1111 1116 1121 1126 1132 1144 1149 1154 1159 1204 1209 1214 1219 1224 1237 1240 1248 2 Fluid Rate 135

300/ 84-325 P or A rate 250/70 275/77 / 91 P/A Top Up# 1116 1138

HR bpm 83 70 83 80 79 74 145 162 97 94 88 77 113 105 102 75 73 64 69 86 SAP mmHg 154 137 129 126 123 124 95 95 119 138 153 150 150 152 151 124 120 119 108 108 DAP mmHg 66 54 54 53 57 55 55 53 65 70 76 74 77 76 77 60 61 58 58 63 MAP mmHg 89 73 71 70 73 71 67 65 79 88 93 92 95 96 96 78 77 72 73 76 o Temp C

SPO2 96 98 97 97 97 97 96 96 96 99 97 97 97 97 98 98 97 94

ET CO2 34 38 48 46 44 41 45 44 44 44 45 45 45 45 39 38 39 RR 0 5 10 10 9 9 9 9 9 9 9 9 9 9 9 9 9 LiDCO 2.37/CI 2.51 2.24/CI 2.3

1.7/ 2.2/ 2.3/2 4.3/4.4/ 3.5/3 3.6/3 2.1/2. 1.73 1.69/1 2.6/ 2.5/2.6/2.5/2 PulseCO 3.5/3.6 .2/2. 4.1 .7 .7 2 /1.7 .66 2.7/ .6 4 5 2.1 3.2/3 3.2/3.6/ 3.2/3. 3.4/3.3/ .4/3. PulsCO IND 3.4 4/3.5 3.5 5 1min 3min 4 min 2min Anesth score 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0

159

Alonzo Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/14/14 Time 0 Time 1240 1243 1248 1253 1258 1313 1328 1343 Score R0 R0 R0 P3 P3 P3 P1 Other Sternal 1342 Stand 1403

PlasmaSample

Alonzo 3min after Pre-Vent End MRI Oct/14/14 fentanyl Na+ 147 148 148 Cl - 119 118 117 K+ 3.5 3.3 3.9 Hb 17.6 15 13.2 pH 7.347 7.215 7.279 PCO2 35.4 53.7 46.8 PaO2 92.4 525 528 HCO3- 18.4 20.6 21.5 ABE -5.2 -7.1 -5.1 Lactate 1.2 0.8 0.5 PCV (%) 50 44 38 TP (mg/dL) 60 58 52 Plasma Sample:

160

Appendix 2.2: Raw data of dog 2

HR RR TEMP Attitude Date:______Oct 17 2013______Before Instrumentation 86 panting 38.5 BAR-relaxed Randomization Dog number: ______7______Time Response Attempts Dog ID#: ______EMLA application Dog Name: ______Baby______Cephalic vein Meloxicam 1305(0.42ml) Dog Weight:______19.5______KG Dorsal pedal artery 1311 no 1 After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______1317____ 176 137 110 88 8.7 Blood Gas:_1312_ 182 panting 146 122 99 4.2/CI5.21 8.5 Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______1323_____ 2.6 Yes No No 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 1326 Blood Gas:______144 panting 165 120 95 NA 7.0 Time Starting Induction: ______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______1331______Mid/Sal Volume:______1 2 3 4 5 6 7 Total dose Intubation score: Injectable Volume y y y y y y y 5ml ______3______Attempt to Intubate y Comments: Relaxed jaw tone Lateral palpebral Medial palpebral Coughing Swallowing Paddling Vocalization 161

POST- Baby IPPV INTO Out of Pre-Reco 5 min 10 min 15 min 5 min 10 min 15 min 20 min 25 min 30 min 35 min 40 min 45 min Oct/17/13 Starts MRI MRI very Intub TIME 1331 1336 1341 1346 1350 1400 1405 1410 1415 1420 1425 1430 1435 1440 1445 1451 1456 Fluid Rate 100 100 100 100 100 100 100 100 100 100

250->27 325->35 P or A rate 275 300 375 375 375 350 325 325 325 325 325 325 325 325 325 5 0 P/A Top Up 2 4 1

12 11 HR bpm 125 121 128 131 112 108 100 95 92 89 99 85 80 85 60 59 66 3 5 10 SAP mmHg 127 120 119 94 88 89 80 79 78 78 72 86 103 97 100 95 90 96 2 DAP mmHg 72 66 64 56 51 52 47 43 47 47 49 49 50 58 56 55 60 55 50 MAP mmHg 90 83 80 72 62 65 58 55 56 55 57 55 60 70 67 65 73 68 65 o Temp C

SPO2 96 99 97 98 98 96 96 98 98 98 98 98 98 98 98 98 98

ET CO2 26 49 51 53 48 45 44 41 40 40 39 39 39 39 38 36 42 RR 15 26 22 8 11 10 10 10 10 10 10 10 10 10 10 12 12 2.26/CI:2 1.53/CI:2. LiDCO 4:7.1 .81 16 PulseCO 0:6.2 7.4 6.5 5.8 5.3 4.8 1.6 1.6 2.2 PulseCO IND 1:7.1 2:7.3 3:7.3 Anesth score 1 1 1 1 1 3 0 0 0 0 0 0 0 0 0 1 0

162

Baby Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/17/13 Time 0 Time 1502 1506 1511 1516 1521 1536 1551 1606 Score R0 R0 R0 P1 P1 P0 P0 Other 1519 1545 stand sternal PlasmaSample

Baby Before Before IPPV Out of MRI Oct/17/13 premed Na+ 148 150 147 Cl - 119 119 118 K+ 3.6 3.2 3.9 Hb 19 16.3 15 pH 7.379 7.256 7.381 PCO2 35.6 52 36.6 PaO2 100 539 496 HCO3- 22.0

ABE -3.9 -3.8 -3.0 Lactate 1.1 1.1 0.5 PCV (%) 54 46 44 TP (mg/dL) 6.2 5.8 5.4 BUN 5-15

163

HR RR TEMP Attitude Date:______Oct 31_2013______Before Instrumentation 150 panting calm Randomization Dog number: ______tx2______Time Response Attempts Dog ID#: ______EMLA application 0730 Dog Name: ______Baby______Cephalic vein 0745 No 1 Meloxicam 0810 Dog Weight:______20______KG Dorsal pedal artery 0820 mild 4 After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______0825_____ Blood Gas:______174 panting 170 145 126 NA 6.4 Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______0826______2.8 Yes No No 1 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: Blood Gas:______139 panting 182 132 123 NA 5.4 Time Starting Induction: ___0837______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______0838______Mid/Sal Volume:______1 2 3 4 5 6 7 Total dose Intubation score: Injectable Volume y y y y y y y 5ml ______1______Attempt to Intubate y y Y(in) Comments: Relaxed jaw tone Lateral palpebral N n n n n n n Medial palpebral y y y y y Y y Coughing y N y Swallowing Y Paddling Vocalization

164

POST- Out Baby IPPV INTO 10 15 20 25 30 35 40 45mi Pre-Reco TIVA extub 5min 5 min 10 min 15 min 5 min of Oct/31/13 Starts MRI min min min min min min min n very stop ation post Intub MRI TIME 0838 0853 0900 0906 0951 0955 1000 1005 1017 1022 Fluid Rate 250/70 325/91- 375/1 400/1 425/1 450/1 P or A rate -275/7 300/84 300/84 stop 350/98 05 12 19 26 7 P/A Top Up 1 1 1 1 13 12 HR bpm 127 127 130 145 125 107 103 104 109 107 116 131 129 127 85 77 73 70 75 113 2 0 10 SAP mmHg 114 117 98 94 72 76 80 82 81 83 99 87 113 139 136 96 92 90 85 100 110 6 DAP mmHg 85 66 56 64 54 51 60 52 55 55 56 65 60 84 106 103 61 59 53 51 61 72 MAP mmHg 97 81 70 77 66 60 69 62 64 64 66 78 70 94 117 113 72 67 64 61 72 83 o Temp C

SPO2 96 98 99 99 99 98 98 97 97 97 96 96 95 95 95 97 97 97

ET CO2 0 36 46 50 48 43 44 44 44 44 45 41 43 45 44 40 38 RR 0 0 33 16 11 11 11 11 11 11 11 11 11 11 11 11 11 2.72/CI 3.62/CI: 2.02/CI:2. LiDCO :3.68 4.89 75 2. 4. PulseCO 4.2 2.6 2.7 3.6 2.3 2.2 2.1 1.8 2.1 2.6 6 7 PulseCO IND 1:6.4 2:6.7 3: Anesth score 1 2 1 2 1 1 1 1 1 2 1 1 1 1 1 1 1 1

165

Baby Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/31/13 Time 0 Time 1005 1017 1047 1102 1117 Score R0 R0 R0 P3 P3 P2 P0 Other 1101 1117 stand sternal PlasmaSample

Baby after Before IPPV Out of MRI Oct/31/13 premed Na+ 147 147 147 Cl - 117 117 116 K+ 3.5 3.2 4.1 Hb 21.2 18.3 14.9 pH 7.422 7.259 7.331 PCO2 35.6 53.9 45.2 PaO2 94.6 499 583 HCO3-

ABE 0.2 -4.4 -1.9 Lactate 1.8 2.3 0.9 PCV (%) 58 53 45 TP (mg/dL) 6.4 6.0 5.8 BUN 5-15 166

HR RR TEMP Attitude Date:______Nov 12__2014______Before Instrumentation 68 panting BAR Randomization Dog number: ______tx3______Time Response Attempts Dog ID#: ______EMLA application 0900 Dog Name: ______Baby______Cephalic vein No 1 Meloxicam 0945; 0.4 ml Dog Weight:______20______KG Dorsal pedal artery n 1 After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______1025_____ Blood Gas:______140 16 142 105 99 NA 2.7 Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______1023______2.7 no No No 0 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 110 127 108 86 2.1 Blood Gas:______95 184 129 104 2.1 100 28 190 137 112 3.63/CI:4.5 2.5 Time Starting Induction: ___1040______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______Mid/Sal Volume:______1 2 3 4 5 6 7 Total dose Intubation score: Injectable Volume Y ______0______Attempt to Intubate Y Comments: Relaxed jaw tone Y Lateral palpebral N Medial palpebral Y Coughing y Swallowing Paddling Vocalization

167

Out Baby POST-I IPPV INTO 10 15 20 25 30 35 40 45mi Pre-Reco TIVA extub 5min 5 min 10 min 15 min 5 min of Nov/12/13 ntub Starts MRI min min min min min min min n very stop ation post MRI TIME 1041 1046 1051 1056 1100 1110 1115 1155 1204 1209 1213 1221 1226 Fluid Rate 250/70 300/84 325/9 P or A rate -275/7 -325/9 350/98 1 7 1 P/A Top Up 2 1 2 12 12 HR bpm 96 115 121 122 139 138 138 127 127 122 122 111 99 95 62 60 52 75 72 4 5 10 10 12 SAP mmHg 136 108 106 96 69 75 66 71 76 79 82 82 85 125 115 124 121 5 3 Line 0 DAP mmHg 67 57 58 59 56 54 40 loose 45 39 39 42 45 46 45 46 65 57 53 58 64 MAP mmHg 88 72 72 71 69 67 50 56 48 49 52 55 57 55 57 81 72 71 74 78 o Temp C 36.4

SPO2 99 99 99 99 99 97 97 97 97 98 99 99 99 99 99 97 97

ET CO2 4 44 56 54 46 39 35 36 38 35 35 35 35 38 40 37 RR 0->5 7 4 7 11 7 7 7 7 7 7 7 7 7 8 8 2.39/2. 1.57/CI:1. LiDCO 96 94 8. 2. PulseCO 5.3 8.3 8 2.4 1.2 1 1.1 2 2.1 7 5 PulseCO IND 6.5 6 7.4 Anesth score 3 1 1 1 1 3 0 0 0 0 0 0 0 0 0 1 1

168

Baby Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Nov/12/13 Time 0 Time 1213 1221 1226 1231 1236 1251 1306 1321 Score R0 R0 R2 P3 Other Sternal Stand 1304 1321 PlasmaSample

Baby after Before IPPV Out of MRI Nov/12/13 premed Na+ 150 151 150 Cl - 3.7 3.2 3.4 K+ 120 121 120 Hb 17.6 16.1 14.2 pH 7.338 7.206 7.339 PCO2 41.6 61.1 39.4 PaO2 77.5 518 607 HCO3- 21.3 23.3 20.9 ABE -3.3 -5.8 -4.3 Lactate 0.7 0.6 0.5 PCV (%) 48 46 41 TP (mg/dL) 64 58 56 BUN 5-15 169

HR RR TEMP Attitude Date:______Oct 7 2014______Pre-Instrumentation 108 panting 39.4 nervous Randomization Dog number: ______Time Response Attempts Dog ID#: ______EMLA application 0731 Dog Name: ______Baby______Cephalic vein 0752 minimal ` Meloxicam 0755 Dog Weight:______23______KG Dorsal pedal artery 0805 After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______Blood Gas :______181 42 143 128 116 3.0/2.8/2.7 Fentanyl Admin Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______0825______3.2 Yes No No 2 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 148 panting 186 131 107 4.2/3.9/4.5

BloodGas:______3.8/3.9/4.1/ 4/4.5/4.4/5. 141 panting 201 141 119 4.89/CI 6.07 2/5.3 Time Starting Induction: ______0835______P/A Vol: ______Time: Time: Time: Time:083 Time: Time: Time Intubation: ____0837______0835 0836 0836 7 Mid/Sal Vol :______1 2 3 4 5 6 Total dose Intubation score: Injectable Volume ______1______Attempt to Intubate X Comments Relaxed jaw tone Lateral palpebral Medial palpebral Coughing Swallowing Paddling Vocalization

170

5 min POST- Out Baby 5 10 IPPV INTO Pre-Reco TIVA post 15 min 5 min 10 min 15 min 20 min 25 min 30 min 35 min 40 min 45 min of Oct/07/14 min min Starts MRI very OFF extub Intub MRI ation TIME 0837 0842 0847 0852 0859 0940 0945 0950 0955 1000 1005 1010 1015 1020 1025 1031 1036 1039 1050 Fluid Rate

275/7 300/8 P or A rate 250/70 325/91 7 4 P/A Top Up# 0843 0901

HR bpm 151 136 154 162 161 158 111 94 96 95 102 107 125 121 120 125 89 81 90 89 126 SAP mmHg 155 115 104 102 106 100 83 81 78 75 74 76 101 109 110 111 111 93 90 89 107 DAP mmHg 90 62 60 60 60 60 55 51 47 46 44 46 66 73 71 75 56 53 51 53 59 MAP mmHg 109 79 76 76 76 74 66 60 59 55 55 56 78 86 84 87 72 64 63 62 74 o Temp C

SPO2 97 98 98 97 97 96 97 98 97 98 97 97 97 97 97 95 96 96 94

ET CO2 54 54 46 29 36 37 39 40 41 43 44 43 43 34 36 38

RR 0 0 0 9 10 16 10 10 10 10 10 10 10 10 10 10 10 10 1.87/CI LiDCO 3.3/CI 3.76 2.34 1.4/1. 1.8 6.7/6.6 1.1 2.7/2.8 6.2/6. 6.4/6. 6.2/ 3/2. 2.5/2. 51/1. /1. 1.7/1. PulseCO /6.2/6. 7/1 /2.8/2. 3/6.4 5 6.3 9 7/2.9 56/1. 7/1 8 3 .3 9 61 .7 6.4/6. 6.4/6.3 6.1/6.2 6.7/6. 8/6.6./ PulsCO IND /6.5 1min 4/6.5 6.3 4 min 2min 3min Anesth score 0 1 0 0 1 0 0 0 0 0 0 0 1 0 0 1 0

171

Baby Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/07/14 Time 0 Time 1039 1045 1050 1055 1100 1115 1130 1145 Score R0 R0 R0 P3 P3 P1 P0 Other Sternal 1132 Stand 1145

PlasmaSample

Baby 3min after Pre-Vent End MRI Oct/07/14 fentanyl Na+ 150 151 150 Cl - 119 119 118 K+ 3.7 3.3 3.9 Hb 20.7 19.3 15.4 pH 7.408 7.230 7.335 PCO2 33.2 55.7 42.4 PaO2 102 362 449 HCO3- 20 22.1 22.3 ABE -2.0 -6.1 -3.4 Lactate 1.9 3.2 1.5 PCV (%) 61 56 45 TP (mg/dL) 66 64 56 Plasma Sample:

172

HR RR TEMP Attitude Date:______Oct 15_2013______Pre-Instrumentation 144 36 38.6 nervous Randomization Dog number: ______Time Response Attempts Dog ID#: ______EMLA application 0713 Dog Name: ______Baby______Cephalic vein 0728 Meloxicam 0732 Dog Weight:______23______KG Dorsal pedal artery 0740 After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______Blood Gas :______187 44 207 134 111 5.3/5.7/4.6 Fentanyl Admin Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______0754______3.2 Yes/No Yes/No Yes/No 2 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 141 Panting 201 144 122 3.7/3.8/4

BloodGas:______4.3/4.2/4.1/ 3.8/3.9/3.7/ 141 panting 202 144 117 3.87/CI 4.8 3.6/3.5 Time Starting Induction: ______0805______P/A Vol: ______Time: Time: Time: Time: Time: Time: Time Intubation: ______0______0805 0806 0806 0807 Mid/Sal Vol :______1 2 3 4 5 6 Total dose Intubation score: Injectable Volume ______Attempt to Intubate x Comments Relaxed jaw tone Lateral palpebral Medial palpebral Coughing Swallowing Paddling Vocalization

173

5 min POST- Out Baby IPPV INTO Pre-Recove TIVA post 5 min 10 min 15 min 5 min 10 min 15 min 20 min 25 min 30 min 35 min 40 min of Oct/15/14 Starts MRI ry OFF extuba Intub MRI tion TIME 0807 0812 0817 0822 0829 0838 0843 0848 0853 0858 0903 0908 0913 0918 0925 0930 0935 0945 Fluid Rate

P or A rate 250/70 275/77 300/84

P/A Top Up# 0819 0838

HR bpm 130 125 138 146 135 138 127 120 113 111 110 101 101 101 107 73 70 69 69 134 SAP mmHg 141 114 107 104 104 92 85 79 83 83 84 82 84 84 87 109 100 100 99 107 DAP mmHg 77 63 62 62 60 69 46 48 50 50 50 49 50 50 52 56 53 51 51 64 MAP mmHg 97 82 78 77 75 57 62 58 60 60 60 58 59 59 62 70 66 64 63 77 o Temp C

SPO2 97 97 97 98 98 98 98 98 98 98 98 98 99 99 98 97 97

ET CO2 39 41 51 60 53 45 44 44 44 44 43 44 44 44 40 39 37 RR 0 25 17 6 9 12 12 12 12 12 12 12 12 12 12 12 12 1.89 CI LiDCO 1.81 CI 2.25 2.35 4.9/5/ 4.6/4 1. 0.9/ 1.8/1 3.7/3.8/ 4.7/4.8/ 1.7/1. PulseCO 5.1/5.2 .7/4. 8/ 0.9 0.7/ .6/1. 2.1 3.9 4.9/5 8 /5.3 8 2 0.8 7

3.3/3.4/ 4.5/4. 4.6/4.7/ 4.1/4.2/ PulsCO IND 3.5/3.9 7/4.8 4.9/5 4.5/4.7 1min 3min 4min 2min Anesth score 0 0 1 0 0 1 0 0 0 0 0 0 0 0 1 0

174

Baby Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/15/14 Time 0 Time 0935 0940 0945 0950 0955 1010 1025 1040 Score R0 R0 R0 P3 P2 P1 P0 Other paddling Sternal 1012 Stand 1021

PlasmaSample

Baby 3min after 5 min End MRI Oct/15/14 fentanyl post-intubate Na+ 149 149 147 Cl - 117 117 116 K+ 3.5 3.4 4.1 Hb 19.8 19.1 14.2 pH 7.384 7.201 7.295 PCO2 38.3 63.5 50.6 PaO2 80.8 442 364 HCO3- 21.7 23.4 23.8 ABE -1.4 -5.9 -2.7 Lactate 1.7 2.5 0.7 PCV (%) 56 55 41 TP (mg/dL) 64 64 58 Plasma Sample:

175

Appendix 2.3: Raw data of dog 3

HR RR TEMP Attitude Date:______Oct 16 2013______Before Instrumentation 86 panting 38.4 Rowoy/BAR Randomization Dog number: ______no.4______Time Response Attempts Dog ID#: ______EMLA application 1202 Dog Name: ______Baron______Cephalic vein 1230 no 1 Meloxicam 12:38 0.56ml Dog Weight:______28______KG Dorsal pedal artery 1234 no 1 After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______131 97 87 5.43/CI6.5 Event 8 Blood Gas:_1235_ 108 30 139 90 81 7 5.4 147 15.9 Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______1255______3.9 No No No 2 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: Blood Gas:_1257__ 68 9 166 109 88 5.0 Time Starting Induction: ______1300______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______1301______Mid/Sal Volume:______1 2 3 4 5 6 Total dose 5.6ml Intubation score: Injectable Volume y y y ______2______Attempt to Intubate Y Comments: Relaxed jaw tone Y Lateral palpebral Y Medial palpebral Y Coughing y y Swallowing N Paddling N Vocalization n

176

POST- Out Baron 10 15 IPPV INTO 10 15 20 25 30 35 40 45 50 55 60 Pre-Reco TIVA 5 min 5 min of Oct/16/13 min min Starts MRI min min min min min min min min min min min very OFF Intub MRI TIME 1301 1405 1410 1413 1420 1428

Fluid Rate 140 140 140 140 140 140 140 140 140 140

P or A rate 250 275 300 300 300 325 325 325 325 325 300 300 300 300 300 300 300

P/A Top Up 1 1 1 3

5 HR bpm 70 72 73 75 75 86 90 83 83 94 98 96 96 96 96 56 55 6 1 9 SAP mmHg 155 139 115 94 91 82 90 90 83 84 80 83 77 81 87 123 1 106 7 9 5 5 DAP mmHg 64 76 59 51 55 55 56 55 52 51 48 50 46 48 52 58 55 6 6 6 6 MAP mmHg 93 91 76 64 66 63 66 63 60 60 57 60 55 58 62 73 70 8 9 o Temp C

SPO2 95 95 96 96 96 97 98 98 97 96 95 98 100 100 96 98 97

ET CO2 41 41 42 53 44 35 34 36 39 41 41 43 42 42 42 39 37

RR 0 27 0 22 14 12 12 12 12 12 12 12 12 12 12 13 13

3.222/ 2.21/CI LiDCO CI 3.9 2.67 4.7;3.9 PulseCO 6.5 ;4.2;5; 5.6 4.3 4.3 3.4 3.4 5 Anesth score 1 0 0 0 1 1 0 0 1 0 0 0 0 0 0 2 1

177

Baron Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/16/13 Time 0 Time 1431 1436 1441 1446 1451 1506 1521 1536 Score R0 R0 R0 P0-1 P0 P0 Other 1455 1502 Retrun to sternal stand normal

PlasmaSample

Baron Before Before IPPV Extubation Oct/16/13 premed Na+ 147 148 147 Cl - 118 116 115 K+ 4.3 3.6 3.9 Hb 15.9 14.9 13 pH 7.431 7.252 7.331 PCO2 31.4 53.8 42.1 PaO2 93.4 580 563 HCO3- 22.8 ABE -3.1 0.5 -3.2 Lactate 0.7 -2.8 0.4 PCV (%) 45 45 38 TP (mg/dL) 6.0 5.6 5.6 Plasma Sample: 178

HR RR TEMP Attitude Date:______Oct 30_2013______Before Instrumentation 108 48 38.6 excited Randomization Dog number: ______tx2______Time Response Attempts Dog ID#: ______EMLA application 0822 Dog Name: ______Baron______Cephalic vein 0832 no 1 Meloxicam 0.68ml/0845 Dog Weight:______26______KG Dorsal pedal artery 0842 no 1 After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______0846______Blood Gas:______93 42 160 127 110 NA NA NA NA Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: _____0850______3.8 No No Yes 2 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 75 177 111 87 3.33/CI:3.6 3.2 Blood Gas:______105 42 166 106 86 1 3.1 Time Starting Induction: ______0907______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______0909______Mid/Sal Volume:______1 2 3 4 5 6 Total dose Intubation score: Injectable Volume y y y 2.4 ______1______Attempt to Intubate n y Y(in) Comments: Relaxed jaw tone Lateral palpebral n n N Medial palpebral y y y Coughing y Swallowing y Paddling Vocalization

179

POST- Out Baron 10 IPPV INTO Pre-Recov TIVA extub 5min 5 min 15 min 5 min 10 min 15 min 20 min 25 min 30 min 35 min of Oct/30/13 min Starts MRI ery off ation post Intub MRI TIME 0910 0925 0929 0937 0942 1012 1019 1027 1029 1038 1043 Fluid Rate 325/9 250/70- 375/1054 425/11 450/12 450/12 450/12 450/12 475/13 500/14 500/14 500/14 500/ P or A rate 300/84 1-350/ 500/140 275/77 00/112 9 6 6 6 6 3 0 0 0 140 98 P/A Top Up 3 1 1 1 1 1 HR bpm 140 110 96 122 94 111 90 111 106 105 103 103 104 104 88 79 79 71 83 144 10 SAP mmHg 132 118 107 111 101 102 107 95 99 102 103 101 99 91 95 96 96 99 100 6 DAP mmHg 74 66 60 64 61 58 61 61 56 57 60 62 60 59 61 58 59 56 55 64 MAP mmHg 93 76 75 77 70 70 74 74 68 70 72 73 71 71 69 68 66 66 67 86 o Temp C

SPO2 96 98 98 98 98 98 96 96 96 96 99 99 99 99 95 97

ET CO2 0 41 49 46 43 41 42 45 45 43 43 43 43 42 42 RR 0 7 11 13 11 11 10 11 11 11 11 11 11 11 11 LiDCO 2.6/CI:2.82 PulseCO 5.8 4.7 4.1 4.4 3.2 2.1 1.9 2.4 2.1 2.6 3.3 PulseCO IND 1:4.8 2:4.4 3:4.6

Anesth score 2 2 2 2 1 2 1 1 1 1 2 1 1 1 1 1

180

Baron Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/30/13 Time 0 Time 1029 1038 1043 1048 1053 1108 1123 1138 Score R0 R1 R1 P3 P2 P0 P0 Other Sternal 1112 Stand 1115 PlasmaSample

Baron after Before IPPV Out of MRI Oct/30/13 premed Na+ 145 148 147 Cl - 118 118 115 K+ 3.7 3.2 3.8 Hb 16.9 15.2 13.6 pH 7.360 7.280 7.326 PCO2 39.4 50.7 46.1 PaO2 82.8 563 589 HCO3- 21.2

ABE -2.7 -2.7 -2.3 Lactate 0.8 0.9 0.5 PCV (%)

TP (mg/dL)

BUN 181

HR RR TEMP Attitude Date:______Nov 11 2013______Before Instrumentation 86 40 38.4 BAR Randomization Dog number: ______tx3______Time Response Attempts Dog ID#: ______EMLA application 0720 Dog Name: ______Baron______Cephalic vein no 1 Meloxicam 0.56ml Dog Weight:______28______KG Dorsal pedal artery After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______Blood Gas:______130 40 148 111 96 6.8 Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: _____0835______3.9 No No No 3 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 80 141 99 84 4 Blood Gas:______72 153 91 76 3.9 83 pant 153 101 83 3.8/4.11 3.5 Time Starting Induction: ______0856______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______0857______Mid/Sal Volume:______1 2 3 4 5 6 Total dose Intubation score: Injectable Volume y Y 2.8 ______1______Attempt to Intubate y y Comments: Relaxed jaw tone Y Lateral palpebral y Medial palpebral Coughing Swallowing Paddling Vocalization 182

POST- Out Baron IPPV INTO Pre-Recov TIVA extubati 5min 5 min 10 min 15 min 5 min 10 min 15 min 20 min 25 min 30 min 35 min 40 min of Nov/11/13 Starts MRI ery off on post Intub MRI TIME 0857 0902 0907 0912 0917 0932 0937 1007 1017 1023 1026 1033 1038 Fluid Rate 250/70 P or A rate —300/ 325/91 350/98 375/105 400/112 375/105 400/112 84 P/A Top Up 1 1 2 1 11 11 HR bpm 120 111 102 114 127 120 115 113 113 111 110 102 100 69 67 68 66 67 0 1 SAP mmHg 133 101 105 93 86 70 63 71 71 70 68 68 71 96 96 113 100 98 96 102 DAP mmHg 70 58 56 53 50 45 46 48 46 46 43 42 43 65 66 69 57 57 56 59 MAP mmHg 89 72 70 66 63 55 54 58 57 56 54 53 53 76 77 81 70 68 65 71 o Temp C 37.2

SPO2 95 99 98 99 99 99 99 99 99 99 99 99 99 99 97 96

ET CO2 39 42 53 57 44 34 38 39 39 39 40 40 40 40 43 42 RR 0->18 115 8 8 12 12 10 10 10 10 10 10 10 10 10 10 4.96/5.3 3.91/CI:4.2 LiDCO 7 3 4. PulseCO 4.8 4.2 4.7 5.5 4.4 2.5 2.5 3.9 5.1 5.9 8 PulseCO IND 3.7 4.1 4 Anesth score 2 1 1 1 1 3 0 0 0 0 0 0 0 0 1 1

183

Baron Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Nov/11/13 Time 0 Time 1026 1033 1038 1043 1048 1103 1118 1133 Score R0 R0 R0 P3 P2 P0 P0 Other paddling 1101 1112 stand sternal PlasmaSample

Baron after Before IPPV Out of MRI Nov/11/13 premed Na+ 147 148 146 Cl - 3.7 3.3 3.9 K+ 117 118 115 Hb 17.1 15 13.9 pH 7.342 7.223 7.297 PCO2 41.6 57.2 45.3 PaO2 76.4 510 614 HCO3- 21.6 22.6 22.1 ABE -3 -5.6 -4.7 Lactate 1.1 1.1 0.9 PCV (%) 49 48 40 TP (mg/dL) 60 56 58 BUN 5-15 184

HR RR TEMP Attitude Date:______Oct 8_2014______Pre-Instrumentation 120 panting 38.4 calm Randomization Dog number: ______Time Response Attempts Dog ID#: ______EMLA application 0730 Dog Name: ______Baron______Cephalic vein Meloxicam 0752 Dog Weight:______28.4______KG Dorsal pedal artery After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______Blood Gas :______111 panting 136 104 95 2.5/2.3/2.4 Fentanyl Admin Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______0804___ 4 No No Yes 3 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 2/1.9/2.1/2. 68 42 158 102 83 2 BloodGas___0805______65 160 105 88 2.07/CI 2.19 1.4 Time Starting Induction: ______0816______P/A Vol: ______Time:081 Time:0816 Time: Time: Time: Time: Time Intubation: ______0816______6 Mid/Sal Vol :______1 2 3 4 5 6 Total dose Intubation score: Injectable Volume X ______0______Attempt to Intubate Comments Relaxed jaw tone Lateral palpebral Medial palpebral Coughing X Swallowing Paddling Vocalization X

185

5 min POST- Out TIV Baron IPPV INTO post 5 min 10min 15 min 5 min 10 min 15 min 20 min 25 min 30 min 35 min 40 min of Pre-Recovery A Oct/08/14 Starts MRI extuba Intub MRI OFF tion 094 TIME 816 821 826 0831 0837 0852 0857 0902 0907 0912 0917 0922 0927 0932 0941 0946 0959 9 Fluid Rate

275/7 P or A rate 250/70 300/84 325/91 300/84 7 P/A Top Up# 0828 0838

HR bpm 77 67 83 94 99 102 104 104 103 104 105 103 103 117 99 72 62 62 60 80 SAP mmHg 136 98 92 88 89 90 85 73 71 68 69 69 69 86 96 105 102 105 106 109 DAP mmHg 75 55 53 51 51 52 41 45 44 42 42 42 42 54 64 61 58 59 70 75 MAP mmHg 90 66 63 62 62 64 62 55 53 52 52 51 52 66 73 73 69 71 58 62 o Temp C

SPO2 96 97 96 95 98 98 98 98 98 98 97 97 97 98 98 98 97 95

ET CO2 19 16 19 47 40 41 40 41 41 40 40 42 41 39 35 34

RR 0 0 0 0 11 10 10 10 10 10 10 10 10 10 10 10

3.04/CI LiDCO 2.46/CI2.61 3.22 3.1 1.9/2. 3.1/3/3. 2.1/2.2/ 2.3/2. 3.1 /3. 3/2.8/2 1.7/1 1.7/1. 2/2. PulseCO 2/2.1 1/2.2/ 2/2.9/2. 2.1 4 /3 2/3 .9 .8 8 1 2.3 6 .3 1.7/1. 2.1/2.2 1.7/1.8 1.8/1.9 PulsCO IND 8/1.6 1min 3min 4 min 2min Anesth score 0 0 1 0 1 1 0 0 0 0 0 0 0 0 0 0

186

Baron Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/08/14 Time 0 Time 0949 0954 0959 1004 1009 1024 1039 1054 Score R0 R0 R0 P2 P0 P0 P0 Other

PlasmaSample

Baby 3min after Pre-Vent End MRI Oct/15/14 fentanyl Na+ 146 147 145 Cl - 117 116 116 K+ 4 3.6 4.3 Hb 15.3 15.7 14.5 pH 7.381 7.169 7.312 PCO2 37.6 68.2 45.7 PaO2 80.1 491 479 HCO3- 21.8 23.8 22.9 ABE -2.4 -6.4 -3.6 Lactate 1.1 1.7 1.1 PCV (%) 44 46 42 TP (mg/dL) 56 54 58 Plasma Sample:

187

HR RR TEMP Attitude Date:______OCT 15_2014______Pre-Instrumentation 100 24 good Randomization Dog number: ______Time Response Attempts Dog ID#: ______EMLA application Y Dog Name: ______Baron______Cephalic vein Y Meloxicam 1150, 0,.5ml Dog Weight:______26.5______KG Dorsal pedal artery After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______Blood Gas :______122 28 136 93 77 3.4/3.5 Fentanyl Admin Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______1228_____ 3.6 Yes/No Yes/No Yes/No 2 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 73 12 159 90 72 1.9/2 BloodGas ______60 189 105 84 1.8 60 148 98 74 2.24 CI 2.48 2.3/2.4/2.2 Time Starting Induction: ______1236______P/A Vol: ______Time: Time: Time: Time: Time: Time: Time Intubation: ____1237______1236 Mid/Sal Vol :______1 2 3 4 5 6 Total dose Intubation score: Injectable Volume ______1______Attempt to Intubate x Comments Relaxed jaw tone Lateral palpebral Medial palpebral Coughing Swallowing Paddling Vocalization

188

5 min POST- IPPV Out Baron 10 Pre-Recove TIVA post 5 min 15 min Start 5 min 10 min 15 min 20 min 30 min 35 min of Oct/15/14 min INTO ry OFF extuba Intub s MRI MRI 25 min 40 min tion TIME 1237 1242 1247 1252 1256 1302 1307 1312 1317 1322 1327 1332 1337 1242 1348 1352 1355 1404 Fluid Rate

250/70- 300/ P or A rate 325/91 275/77 84 P/A Top Up# 1240 1247 1302

HR bpm 98 77 93 102 105 101 130 139 141 140 142 142 142 138 136 76 72 73 72 82 SAP mmHg 140 127 114 103 105 100 101 73 81 91 102 104 108 108 107 108 107 108 106 108 DAP mmHg 76 67 61 59 60 55 59 50 54 61 71 71 74 72 73 65 65 63 61 60 MAP mmHg 94 82 75 70 72 70 73 58 64 70 79 82 84 81 82 74 77 74 73 72 o Temp C

SPO2 97 97 98 98 98 99 99 98 98 98 98 98 98 98 98 98 98 95

ET CO2 43 43 47 49 41 34 38 39 40 41 42 42 43 43 42 42 41 RR 7 10 10 7 12 12 12 12 12 12 12 12 12 12 12 12 12 LiDCO 2.49/ CI 2.75 1.98 CI 2.14

2.5/ 1.8/ 3.4/3.5/ 3.3/3. 2.6/2. 1.6/1 1.6/1. PulseCO 2.7/2.8 3.5 2.6/ 1.9/ 2/2.1 2/2.1 3.6 4/3.5 5/2.8 .7 7 2.9 2 2.6/2. 1m3.3/ 3/3.1 2.6/2.7 PulsCO IND 7/2.8 3.2in 2min 4 min 3min Anesth score 1 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0

189

Baron Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/15/14 Time 0 Time 1355 1359 1404 1409 1414 1429 1444 1459 Score R0 R0 R0 P3 P3 P2 P1 Other Sternal/stand 1458

PlasmaSample

Baron 3min after Pre-Vent End MRI Oct/15/14 fentanyl Na+ 146 146 147 Cl - 116 117 115 K+ 3.6 3.4 3.6 Hb 14.9 14.7 13.6 pH 7.351 7.315 7.291 PCO2 39.8 42.2 48.6 PaO2 78.9 525 542 HCO3- 21.1 20.9 23.2 ABE -3.3 -4.7 -3.7 Lactate 0.3 0.3 0.3 PCV (%) 43 42 40 TP (mg/dL) 58 54 54 Plasma Sample:

190

Appendix 2.4: Raw data of dog 4

HR RR TEMP Attitude Date:______OCT/15/2013______Before Instrumentation 60 30 calm Randomization Dog number: ______Time Response Attempts Dog ID#: ______EMLA application 1230 Dog Name: ______bolt______Cephalic vein 1245 calm 1 Dorsal pedal artery 1400 Calm 1 Dog Weight:______KG After 30 min rest Time HR RR SAP MAP DAP LiDCO PulseCO SPO2 Na+ Hgb Blood Gas Taken t:_1405_ 79 12 145/146 94/91 77/68 4.32/CI:4.92 146 13.3 1515 88 150/118 98/85 82/64 4.6 Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______1517______3.6 No No No 2 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO SPO2 Na+ Hgb Time: 1520 135/185/ 74/118/ 58/93/8 2.8/6.1/4.09/ Blood Gas Taken :______44/58/67 24 175 107 0 4.66 146 11.9 Time Starting Induction: _____1524______P/A Volume : ______Time:1526 Time: Time: Time: Time: Time: Time Intubation: ______Mid/Sal Volume:______1 2 3 4 5 6 Total dose Intubation Injectable Volume 2.6ml propofol+1.6ml midazolam score: 1 Attempt to Intubate Y Comments: Relaxed jaw tone Y Lateral palpebral N Medial palpebral Y Coughing N Swallowing N Paddling N Vocalization n

191

POST- Bolt IPPV INTO 10 15 20 25 30 35 40 45 50 5 min 10min 15 min 5 min Pre-Recovery Oct/15/13 Starts MRI min min min min min min min min min Intub TIME 1527 1532 1537 1542 1547 1550 1600 1605 1610 1615 1620 1625 1630 1635 1640 1645 1650 1700 1705 1708 Fluid Rate 0 0 0 0 0 0 130 130 130 130 130 130 130 130 130 130 0 0 0 P or A rate 250 250 275 275 275 275 275 275 250 275 275 275 275 275 275 275 275 275 275 P/A Top Up 0 0 1 0 0 0 2 0 0 0 0 0 0 0 0 0 3 0 0 HR bpm 63 62 57 60 75 89 62 64 67 73 72 70 71 73 74 45 41 40 11 SAP mmHg 121 114 135 105 104 99 101 99 100 97 97 98 116 116 118 8 DAP mmHg 56 56 60 46 60 58 55 55 55 55 54 54 53 65 62 59

MAP mmHg 73 72 74 72 71 68 67 66 66 66 65 65 65 77 72 72

o Temp C 37.6

SPO2 96 96 95 99 98 99 99 99 99 99 99 99 99 99 99 96 96 96

ET CO2 54 53 48 49 41 30 31 31 31 32 32 32 31 31 31 33 33 33 RR 9 12 11 10 10 9 9 9 9 9 9 9 9 9 10 10 10

2.4/C 3.5/CI:3.9 LiDCO I:2.7 8 3 PulseCO 5.4 12.1 3.98 3.6 1.5 1.7 1.6 Anesth score 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

192

Bolt Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/15/13 Time 0 Time 1711 1718 1723 1728 1733 1748 1803 1818 Other sternal 1755 stand Score R0 R0 R0 P2 P1 P1

Bolt Before premed 3 min after fentanyl Before ventilation End of MRI Oct/15/13 Na+ 146 146 147 145 Cl - 115 116 117 115 K+ 3.6 3.5 3.2 3.5 Hb 13.3 11.9 13.5 11.6 pH 7.361 7.352 7.262 7.368 PCO2 36.1 35.7 44.2 34.9 PaO2 98.8 98.1 523 533 HCO3- 19.5 18.9 19.1 19.8 ABE -4.2 -5.1 -7.1 -4.6 Lactate 1.9 1.9 2.4 1.7 PCV (%) 37 35 39 37 TP (mg/dL) 6.2/ BUN 5-15 5.8 5.8 5.8 Plasma Sample: 193

HR RR TEMP Attitude Date:______Oct 28______Before Instrumentation 96 panting 38.6 calm Randomization Dog number: ______tx2______Time Response Attempts Dog ID#: ______EMLA application 0940 Dog Name: ______Bolt______Cephalic vein 0950 no 1 Meloxicam 0.52ml,1140 Dog Weight:______26______KG Dorsal pedal artery 1140 non 5 After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______Blood Gas:______70 22 130 96 77 NA 3.1/2.3 NA NA Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______1153______3.6 No No No 1 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 1200 67 185 108 82 1.9/2.4 Blood Gas:______74 30 191 116 94 2.81/3.20 2.7 Time Starting Induction: ______1204______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______1206______Mid/Sal Volume:______1 2 3 4 5 6 Total dose Intubation score: Injectable Volume y y y ______1______Attempt to Intubate Y(in) Comments: Relaxed jaw tone y y Lateral palpebral Y N Medial palpebral Y y Coughing Y Swallowing Paddling Vocalization

194

POST- Out Bolt 10 IPPV INTO Pre-Reco extuba 5min 5 min 15 min 5 min 10 min 15 min 20 min 25 min 30 min 35 min 40 min of Oct/28/13 min Starts MRI very tion post Intub MRI TIME 1206 1221 1225 1235 1315 1321 1328 1335 1349 Fluid Rate 250/70- 300/84- 325/ 375/ P or A rate 325/91 325/91 325/91 325/91 325/91 325/91 325/91 325/91 325/91 375/105 375/105 375/105 275/77 325/91 91 105 P/A Top Up HR bpm 50 55 62 70 71 84 60 57 60 56 59 67 63 64 61 56 53 51 57 57 13 SAP mmHg 146 142 124 111 115 113 97 93 97 100 99 101 109 137 137 124 111 110 103 1 DAP mmHg 84 83 58 58 58 58 66 57 71 71 67 67 80 85 86 70 73 65 71 90 MAP mmHg 63 63 72 69 71 69 79 67 80 80 75 76 70 97 96 87 86 79 82 95 o Temp C

SPO2 98 99 99 97 98 98 96 97 96 96 96 96 96 96 97 97 97

ET CO2 42 47 49 49 45 44 41 41 40 40 40 40 41 39 38 37 37 RR 0 9 8 8 11 11 11 11 11 11 11 11 11 11 11 9 11 2.58/CI:2.9 2.16/CI:2. LiDCO 4 46 1. PulseCO 3.8 2.8 3.0 2.9 3.1 2.3 5.0 1.5 2.2 2.2 5 PulseCO IND 1:2.7 2:2.9 3:2.6 Anesth score 2 2 1 1 1 2 1 1 1 1 1 1 2 1 1 1

195

Bolt Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/28/13 Time 0 Time 1335 1344 1414 1444 Score R0 R0 R0 P3 P1 P0 P0 Other 1414 sternal 1419 stand PlasmaSample

Bolt after Before IPPV Out of MRI Oct/28/13 premed Na+ 144 146 145 Cl - 118 116 114 K+ 3.8 3.4 4.0 Hb 12.8 14.1 12.5 pH 7.426 7.227 7.326 PCO2 30.1 53.8 42.5 PaO2 95.4 497 547 HCO3- 19.1 21.3 21.8 ABE -3.3 -6.2 -3.8 Lactate 1.5 2.2 2.0 PCV (%) 40 40 36 TP (mg/dL) 6.0 6.0 6.0 BUN 5-15 196

HR RR TEMP Attitude Date:______Nov8_2013______Before Instrumentation 72 20 38.4 BAR Randomization Dog number: ______tx2______Time Response Attempts Dog ID#: ______EMLA application Dog Name: ______Bolt______Cephalic vein Meloxicam Dog Weight:______26______KG Dorsal pedal artery After 30 min rest:______HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Blood Gas :__ 79 20 122 98 76 2.4 Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______1051_____ 3.6 n n n 2 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 70 24 150 93 74 1.9 Blood Gas Taken :__ 95 240 146 97 2.4 168 118 90 4.87/5.54 5.2 Time Starting Induction: ___1101______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: _____1102______Mid/Sal Volume:______1 2 3 4 5 6 Total dose Intubation score:2 Injectable Volume Admin y y y y 2.9 ______Attempt to Intubate y y Comments: Relaxed jaw tone y y Y Lateral palpebral y y Y Medial palpebral y y Y Coughing Swallowing y Paddling Vocalization 197

POST- Bolt IPPV INTO 10 15 20 25 30 35 40 45 50 Out of Pre-Recov TIVA extub 5min 5 min 10 min 15 min 5 min Nov/08/13 Starts MRI min min min min min min min min min MRI ery stop ation post Intub TIME 1103 1108 1113 1118 1120 1128 1133 1217 1221 1226 1230 1237 1242 Fluid Rate 350/98- 250/70- 300/84- 400/11 425/1 375/10 P or A rate 275/77 325/91 2 19 5 P/A Top Up 2 2 1 HR bpm 78 76 89 87 85 106 105 86 82 80 81 78 78 81 79 80 81 66 67 66 72 76 11 14 156 128 124 134 112 113 102 107 103 92 90 93 88 84 87 129 133 132 130 105 SAP mmHg 3 0 DAP mmHg 73 55 56 54 55 56 56 47 60 65 59 56 55 58 57 57 58 70 69 65 64 63 MAP mmHg 93 73 73 70 70 72 72 71 72 76 70 65 64 66 64 65 65 82 82 76 73 75 o Temp C 38.2

SPO2 98 96 96 95 99 99 99 99 97 99 99 99 99 99 99 99 98 99

ET CO2 35 43 46 43 44 40 40 34 36 36 36 35 35 35 35 35 35 34 RR 0 12 7 6 8 8 8 8 8 8 8 8 8 8 8 8 11 11 3.37/3.8 1.97/2.24 LiDCO 4 4. 3.3 3.4 4.6 3.9 3.5 1.5 1.7 1.8 2.5 2.1 PulseCO 3 PulseCO IND 3.3 3.1 3.1 Anesth score 3 3 1 1 1 1 0 0 1 0 0 0 0 0 0 0 1 1

198

Bolt Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Nov/8/13 Time 0 Time 1230 1237 1242 1247 1252 1307 1322 1337 Score R0 R0 R0 P3 P2 P1 P0 Other 1316 sternal1329 stand PlasmaSample

Bolt after Before IPPV Out of MRI Nov/8/13 premed Na+ 146 147 146 Cl - 117 116 116 K+ 3.6 3.3 3.6 Hb 14.3 13.3 12 pH 7.326 7.203 7.345 PCO2 40.3 55.7 38 PaO2 74.9 561 452 HCO3- 20.1 20.8 20.4 ABE -4.6 -7.1 -4.6 Lactate 1.1 1.6 1.2 PCV (%) 40 39 35 TP (mg/dL) 60 60 56 BUN 5-15 199

HR RR TEMP Attitude Date:______Oct 6_2014______Pre-Instrumentation 96 18 39.1 calm Randomization Dog number: ______Time Response Attempts Dog ID#: ______EMLA application 0718 Dog Name: ______Bolt______Cephalic vein 0737 none 1 Meloxicam 0738 Dog Weight:______29.5______KG Dorsal pedal artery Lt none 1 After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______Blood Gas:______109 22 129 92 77 7.4/7.1/7.5 Fentanyl Admin Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______0805____ 4.1 Yes No No 3 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 99 60 174 108 83 7.6 BloodGas Taken 111 182 116 89 9.4/8.8 ______99 170 112 87 4.19 5.6 147 16.9 Time Starting Induction: ______0822______P/A Time:082 Time:0823 Time:0823 Time: Time: Time: Time Intubation: ______0823______Vol: ______2 Mid/Sal 1 2 3 4 5 6 Total dose Intubation score: Vol :______1______Injectable Volume Comments Admin Attempt to Intubate y Relaxed jaw tone Lateral palpebral X Medial palpebral x Coughing X Swallowing Paddling Vocalization

200

POST- 5 min post Bolt 5 15 IPPV INTO 10 15 20 25 30 35 40 45 Out of Pre-Recove TIVA 10 min 5 min extubatio Oct/06014 min min Starts MRI min min min min min min min min MRI ry OFF Intub n TIME 0824 0829 0834 0839 0842 0903 0908 0913 0918 0923 0928 0933 0938 0943 0948 0956 1004 1009 1017 dopa Fluid Rate 145 mine 250/70- 300/ 275/7 250/7 275/77-> P or A rate >275/7 300/84 325/91 84 7 0 300/84 7 0954/095 P/A Top Up# 0825 0833 0843 0850 9 HR bpm 95 94 110 123 150 125 115 113 113 109 109 92 85 77 96 76 71 75 69 93 SAP mmHg 122 123 110 100 73 74 73 62 61 60 60 60 68 71 71 102 96 95 84 92 DAP mmHg 56 62 57 52 47 58 58 50 51 50 44 43 51 50 49 65 55 57 54 65 MAP mmHg 74 77 72 68 58 65 64 56 55 53 52 50 57 56 57 76 66 67 63 74 o Temp C 38 38.1 37

SPO2 97 95 96 97 98 100 100 100 100 100 100 100 99 99 100 97 98 100 96

ET CO2 42 46 52 57 46 33 39 42 43 44 44 44 45 45 46 35 35

RR 0 29 11 6 12 12 10 10 10 10 10 10 10 10 10 12 12

2.25/ LiDCO CI 1.71/CI 1.81

2.38 4.4/4. 0.88/0.84 1.62/1.44/1. PulseCO 4.7 5.6/5.7 2.2 1.8/1.7 1.6/1.8/1.5 5 /0.79 4/2.1 3.9/4/4 4.1/4.2, 4.1,4 PulsCO IND .3 4.1/4.2 3min min 1min 2min 1(prob Anesth score 1 0 1 0 1 lem w/ 0 0 0 0 0 0 0 0 0 2 1

a-line)

201

Baron Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/06/14 Time 0 Time 1009 1012 1017 1022 1027 1042 1057 1112 Score R0 R0 R0 P3 P1 P0 P0 Other Sternal:1042 Stand:1045

PlasmaSample

Baron 3min after Pre-Vent End MRI Oct/06/14 fentanyl Na+ 147 149 146 Cl - 117 116 115 K+ 3.4 3.2 4 Hb 16.8 15.2 12.9 pH 7.381 7.192 7.312 PCO2 35 54.3 45.3 PaO2 68.2 406 437 HCO3- 20.3 19.1 22.2 ABE -3.5 -9.3 -3.5 Lactate 2.4 3.2 1.4 PCV (%) 48 49 38 TP (mg/dL) 60 60 60 Plasma Sample:

202

HR RR TEMP Attitude Date:____Oct 14_2014______Pre-Instrumentation 76 20 39 calm Randomization Dog number: ______Time Response Attempts Dog ID#: ______EMLA application 0730 Dog Name: ______Bolt______Cephalic vein 0745 Meloxicam 0751 Dog Weight:______29.3______KG Dorsal pedal artery Left 2nd After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______Blood Gas :______94 22 159 106 85 2.5/2.6 Fentanyl Admin Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______0844___ 4.1 Yes No No 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 99 21 218 139 108 3.2/3.1 BloodGas Taken 89 19 211 120 89 3.3/3.4 ______90 pant 181 116 87 3.39/Ci 3.6 3.1/3 Time Starting Induction: ______0852______P/A Time: Time: Time: Time: Time: Time: Time Intubation: ______Vol: ______Mid/Sal 1 2 3 4 5 6 Total dose Intubation score: Vol :______Injectable Volume Comments Admin Attempt to Intubate x Relaxed jaw tone Lateral palpebral Medial palpebral Coughing Swallowing Paddling Vocalization 203

5 min POST- Out TIV Bolt 10 IPPV INTO Pre-Recove post 5 min 15 min 5 min 10 min 15 min 20 min 25 min 30 min 35 min 40 min of A Oct/14/14 min Starts MRI ry extuba Intub MRI OFF tion TIME 0853 0858 0903 0908 0915 0929 0934 0939 0944 0949 0954 0959 1004 1009

Fluid Rate

250/70- 325/9 P or A rate 300/84 325/91 300/84 275/77 1 P/A Top Up# 0902

HR bpm 108 89 116 127 129 138 135 131 127 122 119 117 116 115 115 80 82 84 85 110 SAP mmHg 133 128 109 119 110 90 80 78 82 76 81 77 78 78 81 100 102 102 100 101 DAP mmHg 69 63 54 54 52 49 51 51 54 48 50 47 48 49 49 64 60 61 59 63 MAP mmHg 87 80 70 72 69 65 61 61 64 58 60 57 58 59 59 76 70 71 69 73 o Temp C

SPO2 95 98 98 96 97 99 97 97 97 98 96 96 96 96 99 99 96 96

ET CO2 35 47 54 52 48 37 36 37 37 39 40 40 40 45 42 41

RR 0 17 6 7 11 11 11 11 11 11 9 9 9 9 9 9

LiDCO 4.31/CI 4.57 1.77/CI 1.88

4.2/ 4.7/4 6/6.1 3.4/3. 1.5/1. 2.1/2.2/ 3.8/3.9/ 3.6/3.9/ 4.3/ 1.6/1. 1.8/ PulseCO .9/5/ /5.8/ 7/3.8/ 6/1.4/ 2.3/2.7/ 4/4.3 4/4.1 4.4/ 7 1.9 5.1 5.9 4.1 1.6 2.6/2.5 4.5

3m3.4 3.4/3.9/ 3.1/3 3.7/3.3/ /3.7/3. PulsCO IND 3.1 .2/3. 2.9/3.2 8 1min 4 4 min 3min 2min Anesth score 1 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0

204

Bolt Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/14/14 Time 0 Time 1025 1030 1035 1040 1045 1100 1115 1130 Score R0 R0 R0 P3 P2 P1 P0 Other Sternal 1058 Stand 1124

PlasmaSample

Bolt 3min after Pre-Vent End MRI Oct/14/14 fentanyl Na+ 147 149 147 Cl - 116 115 115 K+ 3.5 3.1 3.7 Hb 14.5 13.9 11.2 pH 7.341 7.144 7.266 PCO2 38.1 66.6 47.5 PaO2 83.4 469 447 HCO3- 19.5 21.5 21 ABE -4.5 -8 -5.6 Lactate 1.8 2.3 1.6 PCV (%) 42 40 43 TP (mg/dL) 60 58 54 Plasma Sample:

205

Appendix 2.5: Raw data of dog 5

HR RR TEMP Attitude Date:______Oct/13/13 ______Before Instrumentation 126 36 38.6 excited Randomization Dog number: _____1-______Time Response Attempts Dog ID#: ______EMLA application 0757 Dog Name: ______Chance______Cephalic vein 0805 Calm 1 Dorsal pedal artery 0815 Calm 1 Dog Weight:______26______KG After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO SPO2 Na+ Hgb Time:____0855______5.56 Blood Gas : 0815 112 24 159 105 85 (CI:6.31) NA NA 144 16.8 g/dL Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: ___9:28______3.6 Yes Yes No 1 2 min after premed HR RR SAP MAP DAP LiDCO PulseCO SPO2 Na+ Hgb Time: 5.58 Blood Gas:__NA___ 95 180 142 106 89 (CI:6.35) NA NA 147 14.5 Time Starting Induction: _____0937______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______0939______Mid/Sal Volume: sal1.6ml 1 2 3 4 5 6 Total dose Intubation score: Injectable Volume Y Y Y 1.9 ______3______Attempt to Intubate N N Y Comments: Relaxed jaw tone N Y Y Lateral palpebral Y Y N Medial palpebral Y Y Y Coughing N N N Swallowing N N Y Paddling Y N N Vocalization Y Y N

206

POST- 5 min Chance 10 15 20 IPPV INTO 10 15 20 25 30 35 40 45 50 55 Pre-Recove TIVA 5 min scan Oct/13/13 min min min Starts MRI min min min min min min min min min min ry OFF Intub start 11 TIME 0940 0945 0950 0955 1000 1005 1016 1021 1026 1031 1036 1041 1046 1051 1056 1101 1106 1111 1124 1129 18 Fluid Rate 130 130 130 130 130 130 130 130 130 130 130 130 0 0 0

84->9 98->1 P or A rate 70 77 112 112 112 105 112 105 105 105 98 98 105 105 105 105 105 0 1 05 P/A Top Up 1 0 2 1 2 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 HR bpm 88 77 74 86 78 107 122 97 89 123 113 105 102 96 97 90 90 97 66 54 53 SAP mmHg 139 116 120 116 115 135 67 88 80 86 70 72 74 73 72 76 76 74 118 130

DAP mmHg 63 53 57 53 54 54 45 53 47 49 43 44 44 45 43 44 43 43 48 49

MAP mmHg 80 68 67 66 67 68 59 64 57 65 53 55 55 55 54 54 53 55 60 63 o Temp C 37.5

SPO2 96 97 97 97 96 97 98 98 98 97 99 99 99 98 98 98 99 99 99 98

ET CO2 57 50 51 49 40 35 38 42 40 41 41 41 41 41 40 40 40 41 39

RR 0 0 10 7 6 17 14 10 11 11 12 12 12 12 12 12 12 12 10 10

1.85 LiDCO 2.55/ /2.1 (CO/CI) 2.91 1 PulseCO 0 2 0 0 buckin Anesth score 3 poor 3 poor 2poor 2 poor poor poor 0 0 0 0 0 0 0 0 stable dorsal g @ 1055

207

Chance Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/13/13 Time 0 Time 1129 1137 1142 1147 1152 1207 1222 1237 1307 other Return to Normal normal walking Score R3 R3 R3 P0 P0 P0 P0 P0 PlasmaSam ple

Chance Before premed Before ventilation End of MRI Oct/13/13 Na+ 144 147 146 Cl - 115 117 115 K+ 3.9 3.3 4.0 Hb 16.8 14.5 12.7 pH 7.398 7.218 7.307 PCO2 33.6 53 45.7 PaO2 89.1 533 514 HCO3- 21.9 20.2 21.4 ABE -3.7 -7.0 -4.6 Lactate 1.7 1.5 1.2 PCV (%) 50 43 37 TP (mg/dL) 6.0/ BUN 5-15 5.4 5.2 Plasma Sample: 208

HR RR TEMP Attitude Date:______Oct 28__2013______Before Instrumentation 108 30 38.7 calm Randomization Dog number: ______Time Response Attempts Dog ID#: ______tx2______EMLA application 0729 Dog Name: ______Chance______Cephalic vein 0750 no 2 Meloxicam 0.52/0751 Dog Weight:______26______KG Dorsal pedal artery 0810 After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______Blood Gas:______120 30 116 94 82 NA 4 Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______0818___ 3.6 No No No 0 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 78 152 106 84 3.4 Blood Gas:______139 18 182 135 106 13.1/11 Time Starting Induction: ______0830______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______0832______Mid/Sal Volume:______1 2 3 4 5 6 Total dose Intubation score: Injectable Volume Admin y y ______1______Attempt to Intubate Y(in) Comments: Relaxed jaw tone Y Lateral palpebral N Medial palpebral N Coughing N Swallowing N Paddling N Vocalization n 209

POST- IPPV Out Chance INTO 5 10 15 20 25 30 35 40 45 50 55 60 Pre-Reco extub 5min 5 min 10 min 15 min Start of Oct/28/13 MRI min min min min min min min min min min min min very ation post Intub s MRI TIME 0832 0847 0855 0905 1005 1012 1020 1034 Fluid Rate 325/ 350/ 375/ 400/ 425/ 450/ P or A rate 250/70 275/77 300/84 91 98 105 112 119 126 P/A Top Up 1 1 2 1 10 12 7 HR bpm 91 80 93 96 134 80 79 77 76 69 69 72 75 79 86 137 143 150 107 90 97 91 1 8 5 1 11 11 10 SAP mmHg 110 97 113 78 85 88 87 95 96 98 99 98 100 132 151 160 168 103 0 89 97 91 3 8 4 3 5 DAP mmHg 60 61 58 58 57 53 50 59 59 55 60 58 58 60 61 64 85 98 107 107 65 53 60 60 8 7 MAP mmHg 71 73 72 72 73 69 60 66 62 64 70 68 69 70 71 74 100 116 126 124 77 64 71 70 0 o Temp C

SPO2 95 97 97 97 97 99 99 96 97 95 97 95 96 96 96 95 96 96 99 99

ET CO2 15 27 22 55 43 45 45 42 41 40 39 39 39 42 42 42 33 38 38 RR 0 0 0 0 11 10 10 10 10 10 10 10 10 10 10 10 10 10 11 11 3.15/CI: 3.22/CI: LiDCO 2.69/CI: 3.59 3.66 4. 5. 1. PulseCO 7.8 8.0 9.2 5.9 2.4 1.6 2.2 2.6 2.5 6 4 5 PulseCO IND 1:7.7 2:2.6 3:4.7 Anesth score 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 1 1 1

210

Chance Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/28/13 Time 0 Time 1023 1034 1104 1134 Score R0 R0 R2 Very Very 1115 stop 1134 1140 dyphoric dysphoric whinning sternal stand/ambulate Other PlasmaSample

Chance after 5 min Before Out of Oct/28/13 premed post-intubation IPPV MRI Na+ 144 145 146 144 Cl - 117 117 117 118 K+ 4.1 3.5 3.6 4.2 Hb 16.4 17.7 15.2 14.6 pH 7.333 7.255 7.142 7.332 PCO2 39.7 46.6 67.5 37.7 PaO2 89.4 247 480 610 HCO3- 20.1 20.7 22.6 19.6 ABE -4.3 -6.2 -7.7 -5.6 Lactate 1.0 1.4 1.1 1.1 PCV (%) 48 45 43 TP (mg/dL) 6.0 5.6 5.4 BUN 5-15 211

HR RR TEMP Attitude Date:______Nov8_2013______Before Instrumentation 114 18 38.2 Randomization Dog number: ______tx3______Time Response Attempts Dog ID#: ______EMLA application 0730 Dog Name: ______Chance______Cephalic vein 0740 Meloxicam 0810 Dog Weight:______26______KG Dorsal pedal artery 0800 1 After 30 min rest: HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Blood Gas :__ 80 18 145 112 97 6.1 Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______0834_____ 3.6 y n n 2 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 98 24 148 98 83 5.7 Blood Gas Taken :__ 92 20 181 104 89 4.2/4.79 4.7 92 pant 180 113 91 Time Starting Induction: ___0844______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______0848______Mid/Sal Volume:______1 2 3 4 5 6 Total dose Intubation score: Injectable Volume y y y y Y 6.8 ______3______Attempt to Intubate Y Comments: Relaxed jaw tone Lateral palpebral N Medial palpebral y Coughing Swallowing Paddling Vocalization 212

POST- Chance IPPV INTO 10 15 20 25 30 35 40 45 50 Out of Pre-Reco TIVA extub 5min 5 min 10 min 15 min 5 min Nov/08/13 Starts MRI min min min min min min min min min MRI very stop ation post Intub TIME 0848 0853 0858 0903 0906 0915 0920 1000 1010 1018 1022 1032 1037 Dopa: Fluid Rate 7 7 5 5 3 5 275/77- P or A rate 250/70 275/77 300/87 P/A Top Up 1 1 1 1 HR bpm 71 62 70 74 77 98 105 96 92 92 93 85 75 66 63 76 58 54 55 81 89 SAP mmHg 111 100 96 94 95 72 69 70 68 68 71 72 84 98 89 80 98 88 81 105 116 DAP mmHg 51 48 46 46 46 40 35 40 34 38 39 38 45 47 41 40 49 45 41 53 62 MAP mmHg 67 61 59 57 58 52 44 50 49 47 47 48 54 58 51 49 62 57 52 67 77 o Temp C 37.1 35.6

SPO2 97 97 97 97 97 97 96 96 96 96 96 96 98 98 98 98 98

ET CO2 33 37 59 33 334 34 38 38 37 38 39 40 39 38 38 RR 0 0 0 0 12 10 10 8 8 8 8 8 8 8 8 11 11 2.47/2.8 LiDCO 2 4. PulseCO 4.7 3.1 3.7 2.8 3.1 1.6 1.6 1.9 2.6 3 1 PulseCO IND 3.4 3.7 3.7 Anesth score 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 1 1

213

Chance Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Nov/08/13 Time 0 Time 1022 1032 1037 1042 1047 1102 1117 1132 Score R0 R2 R2 P2 P2 P1 Other 1047sternal 1115stand PlasmaSample

Chance after Before IPPV Out of MRI Nov/08/13 premed Na+ 144 146 144 Cl - 115 116 115 K+ 3.8 3.5 3.8 Hb 15.5 15.6 12.6 pH 7.35 7.216 7.308 PCO2 41.6 59.3 41.8 PaO2 77.1 466 610 HCO3- 22 23.1 20.8 ABE -2.5 -5.5 -5.2 Lactate 0.9 1.1 0.9 PCV (%) 45 45 36 TP (mg/dL) 54 54 50 BUN 5-15 214

Appendix 2.6: Raw data of dog 6

HR RR TEMP Attitude Date:______Oct/16/2013______Before Instrumentation 102 24 nervous Randomization Dog number: ______no.3______Time Response Attempts Dog ID#: ______EMLA application 0735 Dog Name: ______Hunter______Cephalic vein 0817; 0825 none 3 Meloxicam y Dog Weight:______24______KG Dorsal pedal artery 0851 mild 4 After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______0905____ 144 99 75 5.26/ Blood Gas Taken t:0855_ 119 151 101 88 CI6.36 5.2 142 18.3 Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______0914_____ 3.4 Yes No No 2 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 0925 180 120 96 6.94/ 6.9 Blood Gas Taken :__0916_ 119 panting 182 119 102 CI:8.40 6.2 145 17.4 Time Starting Induction: ______0932______P/A Volume : __1.5_____ Time:0933 Time:0933 Time: Time: Time: Time: Time Intubation: ______0933______Mid/Sal Volume:___1.4___ 1 2 3 4 5 6 Total dose Intubation score: Injectable Volume Admin y y 1.8ml ______2______Attempt to Intubate Y Comments: Relaxed jaw tone Y Lateral palpebral N Medial palpebral Y Coughing Y Swallowing N Paddling N Vocalization n

215

POST- Hunter IPPV INTO Out of Pre-Recove 5 min 10 min 15 min 5 min 10 min 15 min 20 min 25 min 30 min 35 min 40 min 45 min Oct/16/13 Starts MRI MRI ry Intub 105 TIME 0935 0940 0945 0950 0953 1000 1008 1013 1018 1023 1028 1033 1038 1043 1048 1054 1106 4 Fluid Rate 0 0 0 0 0 120 120 120 120 120 120 120 120 120 120 120

P or A rate 70 77 84->91 91 91 84 84 84 84 84 84 84 84 84 84

P/A Top Up 1 2

11 HR bpm 120 107 101 99 124 109 88 104 89 89 83 86 84 93 61 61 58 5 10 SAP mmHg 109 116 108 93 85 78 86 85 80 88 84 82 81 86 105 102 101 3 DAP mmHg 60 66 59 58 53 52 53 57 51 52 56 55 53 54 53 63 62 59 MAP mmHg 75 81 73 72 68 68 62 67 63 60 65 63 61 65 63 76 72 65 o Temp C

SPO2 96 95 95 97 97 97 100 100 100 100 100 100 100 100 100 100

ET CO2 29 43 39 47 44 37 43 31 32 35 33 34 34 34 35 34 34 RR panting 28 21 17 10 8 8 8 8 8 8 8 7 7 13 13 13 3.39/ LiDCO 1.88/CI:2.28 CI:4.1 5. 3. PulseCO 6.1 5.9 5.4 3.6 1.5 1.4 3 2 Anesth score 0 0 2 1 1 1 0 0 0 0 0 1 0 0 0 0

216

Hunter Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/16/13 Time 0 Time 1110 1112 1117 1122 1127 1142 1157 1212 Score Ben R2 R3 R3 P0 P0 P0 P0 Melissa 0 2 3 1 0 0 0 Other Calm Ambulate between 30-45 PlasmaSample

Hunter Before 3 min after Before IPPV Extubation Oct/16/13 premed fentanyl Na+ 142 145 146 146 Cl - 114 116 115 115 K+ 3.9 3.8 3.5 3.8 Hb 18.3 17.4 16.5 13.6 pH 7.398 7.386 7.292 7.371 PCO2 35 37.3 47.9 38.8 PaO2 103 79.8 527 563 HCO3- 20.7 22.9

ABE -2.1 -2.5 -3.2 -2.5 Lactate 0.9 0.7 1.2 0.5 PCV (%) 53 50 47 40 TP (mg/dL) 6.0 5.6 5.6 5.0 BUN 5-15 Plasma Sample: 217

HR RR TEMP Attitude Date:______Oct 30_2013______Before Instrumentation panting 38.6 calm Randomization Dog number: ______tx2______Time Response Attempts Dog ID#: ______EMLA application 0720 Dog Name: ______Hunter______Cephalic vein 0730 no 1 Meloxicam 0741/0.48ml Dog Weight:______KG Dorsal pedal artery 0749 no 1 After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______Blood Gas:______60 panting 118 94 76 NA 1.9 NA NA Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: ____1103______3.4 No No No 0 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: Blood Gas:______115 panting Time Starting Induction: ______1112______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______1114______Mid/Sal Volume:______1 2 3 4 5 6 7 Total dose Intubation score: Injectable Volume Admin y y y y y y y 4.2 ______3______Attempt to Intubate Y(in) Comments: Relaxed jaw tone Lateral palpebral y y Y n N Medial palpebral y y y y Y Coughing y Swallowing Paddling Vocalization 218

POST- Hunter IPPV INTO Out of Pre-Recov TIVA extuba 5min 5 min 10 min 15 min 5 min 10 min 15 min 20 min 25 min 30 min 35 min 40 min Oct/30/13 Starts MRI MRI ery stop tion post Intub TIME 1114 1129 1132 1145 1225 1228 1236 1238 1258 Fluid Rate 325/91- 400/11 425/11 400/11 400/11 P or A rate 250/70 300/84 375/105 350/98 2 9 2 2 P/A Top Up HR bpm 105 100 94 86 87 105 86 87 91 98 102 105 105 103 108 86 70 70 83 121 141 11 10 SAP mmHg 124 124 116 105 95 94 97 98 107 108 123 163 163 106 102 100 93 108 128 2 9 DAP mmHg 68 65 59 56 57 57 50 60 59 60 66 68 81 97 95 58 55 53 52 64 79 MAP mmHg 84 80 74 71 69 69 69 70 69 71 77 79 92 115 113 71 67 68 66 78 96 o Temp C

SPO2 98 98 98 99 96 96 97 96 96 96 98 97 97 97 98 97

ET CO2 0 33 48 46 41 41 42 42 42 42 42 43 43 42 38 36 RR 0 0 15 23 11 11 11 11 11 11 11 11 11 11 11 11 3.05/CI: 2.37/CI:2.8 LiDCO 3.7 7 4. PulseCO 4.3 4.4 4.3 3.1 3.0 2.3 1.5 2.1 3.0 3.0 5.8 1 PulseCO IND 1:3.8 2:4.5 3:4.0 Anesth score 1 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1

219

Hunter Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/30/13 Time 0 Time 1238 1257 1312 1327 1357 Score R2 R3 R3 dysphoric dysphoric P0 P0 Other 1314 1327 stand sternal PlasmaSample

Hunter after Before IPPV Out of MRI Oct/30/13 premed Na+ 147 147 146 Cl - 119 118 118 K+ 3.6 3.4 3.6 Hb 16.8 16.5 14.4 pH 7.382 7.274 7.348 PCO2 37.9 49.8 40.5 PaO2 79.8 556 575 HCO3- 22.8 22.0 21.6 ABE -2.4 -4.6 -3.2 Lactate 0.5 0.9 0.5 PCV (%) 48 47 40 TP (mg/dL) 5.4 5.4 4.8 BUN 5-15 220

HR RR TEMP Attitude Date:______Nov11_2013______Before Instrumentation 90 pant 38..9 calm Randomization Dog number: ______tx3______Time Response Attempts Dog ID#: ______EMLA application 0740 Dog Name: ______Hunter______Cephalic vein n Meloxicam 1140 Dog Weight:______24.5______KG Dorsal pedal artery After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______Blood Gas Taken t:__ 110 20 169 127 107 7.9 Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______1140______3.6 y n n 0 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 104 pant 215 151 112 7.3 Blood Gas Taken :__ 128 133 122 119 5.57/6.04 6.3 Time Starting Induction: ___1145______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______Mid/Sal Volume:______1 2 3 4 5 6 7 Total dose Intubation score: Injectable Volume Admin y y y y y y Y 6.7 ______2______Attempt to Intubate y Y Comments: Relaxed jaw tone Lateral palpebral y y y y y N n Medial palpebral y y y y y y Y Coughing y Swallowing y Paddling Vocalization

221

POST- Hunter IPPV INTO 10 15 20 25 30 35 40 45 Out of Pre-Recov TIVA extub 5min 5 min 10 min 15 min 5 min Nov/11/13 Starts MRI min min min min min min min min MRI ery stop ation post Intub 12 12 TIME 1152 1157 1202 1220 1233 1238 1243 1248 1253 1258 1303 1308 1313 1318 1323 1330 1334 1337 1342 07 24 Fluid Rate 300/84- 250/70- 375/10 P or A rate 325/91- 275/77 5 350/98 P/A Top Up 1 1 1 HR bpm 88 89 92 92 88 86 87 85 74 78 84 86 74 67 69 59 82 58 59 59 74 64 11 10 10 10 SAP mmHg 139 142 125 94 90 86 85 85 88 98 114 113 100 137 138 135 124 126 4 5 9 0 DAP mmHg 60 56 52 53 48 47 44 48 46 42 43 42 43 44 45 47 45 52 56 55 57 55 MAP mmHg 81 74 69 67 60 61 59 62 57 56 55 54 54 58 62 63 60 69 72 70 71 73 o Temp C 38.3

SPO2 97 96 97 98 98 98 96 99 99 99 99 99 99 99 99 97 97

ET CO2 35 36 49 54 54 46 37 39 38 38 37 37 37 37 39 41 40 RR 0 35 15 6 9 10 10 10 10 10 10 10 10 10 10 9 10 LiDCO 1.85/2.18 8. 3. PulseCO 9.1 7.3 8.1 6.7 3.2 1.9 1.6 1.8 2.1 1.8 2 4 Anesth score 2 2 1 1 1 1 0 0 0 0 0 0 0 0 0 1 1

222

Hunter Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Nov/11/13 Time 0 Time 1334 1337 1342 1347 1352 1407 1422 1437 Score R0 R0 R2 P1 P1 P1 Other 1345 1421 stand sternal PlasmaSample

Hunter after Before IPPV Out of MRI Nov/11/13 premed Na+ 147 147 145 Cl - 118 118 116 K+ 3.7 3.5 4.2 Hb 17.2 16.3 14.1 pH 7.366 7.209 7.264 PCO2 37.9 58.5 50.5 PaO2 92.7 551 497 HCO3- 22.1 22 22.2 ABE -3.4 -6.2 -4.8 Lactate 1.1 1.7 0.6 PCV (%) 50 47 40 TP (mg/dL) 58 58 52 BUN 5-15 223

Appendix 2.7: Raw data of dog 7

HR RR TEMP Attitude Date:_____Oct 23 2013______Before Instrumentation 120 36 calm Randomization Dog number: ______9______Time Response Attempts Dog ID#: ______EMLA application 0720 Dog Name: ______Lucky______Cephalic vein 0740 no 1 Meloxicam 0755, 0.5ml Dog Weight:______25.5______KG Dorsal pedal artery 0750 no 1 After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:_____0805______Blood Gas Taken t:______96 24 147 118 99 na na Na na Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______0815______3.6 Yes No No 2 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 0827 55 181 125 108 2.33/CI:2.7 Blood Gas Taken :__0817_ 59 48 152 119 106 5 3.1; 2.3 144 15.4 Time Starting Induction: ______0835______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______0838______Mid/Sal Volume:______1 2 3 4 5 6 7 Total dose Intubation score: Injectable Volume Admin y y y y y y y 6.8ml ______3______Attempt to Intubate y Comments: Relaxed jaw tone Lateral palpebral Medial palpebral Coughing Swallowing Paddling Vocalization 224

POST- Lucky IPPV INTO Out of Pre-Recover 5 min 10 min 15 min 5 min 10 min 15 min 20 min 25 min 30 min 35 min 40 min 45 min 50 min Oct/23/13 Starts MRI MRI y Intub TIME 0838 0843 0848 0853 0901 0910 0915 0920 0925 0930 0935 0940 0945 0950 0955 1007 1012 Fluid Rate 130 130 130 130 130 130 130 130 130 130

300/ 275/ 275/ 275/ 275/ 275/ 275/ P or A rate 250 275 300 300 300 300 300 300 D:4.5 D:4.5 D:7 D:10 D:10 D:7 D:7 P/A Top Up 1 1 2

HR bpm 81 81 85 96 96 96 80 92 92 88 86 72 75 65 60 50 68 70 72 SAP mmHg 105 104 99 97 93 90 61 68 71 73 68 60 75 81 101 104 117 98 101 DAP mmHg 68 63 59 60 54 56 36 42 45 45 42 35 43 42 45 48 63 57 63 MAP mmHg 74 75 71 71 68 67 43 50 53 53 49 41 50 51 56 62 75 68 73 o Temp C

SPO2 95 96 97 97 99 96 98 98 98 98 98 99 99 99 99 98 98

ET CO2 42 47 46 49 40 32 34 35 35 35 35 35 35 35 37 45 37 RR 0 6 6 4 10 10 10 10 10 10 10 10 10 10 10 10 10 3.42/ 2.85/CI: LiDCO 3.63/CI:4.28 CI:4.04 3.37 4.7;5.5; PulseCO 2.5 5.4 Anesth score 0 1 1 1 2 1 1 1 1 1 1 1 1 1 1 2 1

225

Lucky Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/23/13 Time 0 Time 1019 1022 1027 1032 1037 1052 1107 1122 Score R0 R0 R0 P3 P3 P1 P1 Other Sternal PlasmaSample and atand and ambulate on 1110

Lucky 5 min pot-fentanyl 5 min post-intubation Before IPPV Out of MRI Oct/23/13 Na+ 144 145 146 145 Cl - 116 116 116 3.6 K+ 4.1 3.7 3.5 116 Hb 15.4 16.7 14.2 13.1 pH 7.394 7.262 7.222 7.283 PCO2 32.6 46.1 50.6 42.8 PaO2 90.6 180 521 511 HCO3- 19.2 19.8 20 20.1 ABE -3.8 -6.7 -7.6 -6.5 Lactate 1 1.8 1.6 1.3 PCV (%) 46 47 40 38 TP (mg/dL) 6.4 6.2 6.0 5.8 BUN 5-15 226

HR RR TEMP Attitude Date:______Nov5_2013______Before Instrumentation 108 24 38.6 calm Randomization Dog number: ______tx2______Time Response Attempts Dog ID#: ______EMLA application 0730 Dog Name: ______lucky______Cephalic vein 0748 no 2 Meloxicam 0750 Dog Weight:______26______KG Dorsal pedal artery 0800 1 After 30 min rest:0810 HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Blood Gas Taken t:__0822 85 24 138 105 92 4.3 Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______0820____ 3.6 No No No 2 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 78 30 147 107 96 1.6 2.3 Blood Gas Taken :__0822 55 26 154 110 100 1.18 1.7 70 159 116 101 1.34 1.6 Time Starting Induction: _____0838______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______Mid/Sal Volume:______1 2 3 4 5 6 Total dose Intubation score: Injectable Volume Admin y Y ______Attempt to Intubate Y Comments: Relaxed jaw tone Lateral palpebral N n Medial palpebral y y Coughing Swallowing Paddling Vocalization 227

POST- Out Lucky IPPV INTO 10 15 20 25 30 35 40 45mi Pre-Reco TIVA extub 5min 5 min 10 min 15 min 5 min of Nov/05/13 Starts MRI min min min min min min min n very stop ation post Intub MRI 0928 0920 TIME 0839 0854 0859 0905 0910 injecti 0950 0953 1000 1005 1012 1017 needle on Fluid Rate 275/77 250/70 P or A rate -300/8 - 4 P/A Top Up 1 1 HR bpm 80 77 83 98 97 96 83 83 84 82 79 79 81 80 86 86 75 60 63 53 57 63 SAP mmHg 114 105 91 87 81 71 74 73 74 74 72 71 72 71 74 78 79 80 87 87 95 94 DAP mmHg 85 74 64 62 58 52 59 56 57 49 56 54 55 54 57 60 62 59 60 61 67 64 MAP mmHg 85 74 64 62 58 52 59 56 57 49 56 54 55 54 57 60 62 59 60 61 67 64 o Temp C 37.4 35.4

SPO2 96 97 99 98 97 98 98 98 97 97 99 99 98 98 98 97 97 97

ET CO2 0-46 47 52 53 44 40 42 42 42 42 42 42 42 42 42 40 37 37 RR 0-6 11 6 15 11 11 11 11 11 11 11 11 11 11 11 11 11 11 2.64/3. LiDCO 1.68/1.91 01 4. 3. PulseCO 1.8 2.6 3.3 3 1.7 1.5 1.6 1.7 1.8 1.7 3 2 Anesth score 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 1 1

228

Lucky Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Nov/05/13 Time 0 Time 1005 1012 Score R0 R0 R0 P3 P2 P1 Other Sternal 1104 stand 1042 PlasmaSample

Lucky after Before IPPV Out of MRI Nov/05/13 premed Na+ 144 146 153 Cl - 117 117 113 K+ 4.1 3.6 4.4 Hb 15.1 15.5 13.1 pH 7.336 7.165 7.264 PCO2 39.6 61.1 44.3 PaO2 78.1 547 606 HCO3- 20.2 21.1 ABE -4.2 -8.5 -602 Lactate 1.9 2.6 1.2 PCV (%) 43 45 39 TP (mg/dL) 66 64 60 BUN 5-15 229

HR RR TEMP Attitude Date:______Nov19_2013______Before Instrumentation 72 28 38.4 calm Randomization Dog number: ______tx3______Time Response Attempts Dog ID#: ______EMLA application 0720 Dog Name: ______lucky______Cephalic vein 0735 no 1 Meloxicam 0735;0.5 ml Dog Weight:______26______KG Dorsal pedal artery 1 After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Blood Gas Taken t:__ 72 28 139 111 97 2.5 Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______0820____ 3.6 No No No 3 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 51 24 117 89 76 1.9 Blood Gas Taken :__0824 164 120 104 1.5 155 116 102 1.94 1.3 Time Starting Induction: _____0833______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______Mid/Sal Volume:______1 2 3 4 5 6 Total dose Intubation score: Injectable Volume Admin y Y 1.7 ______0______Attempt to Intubate Y Comments: Relaxed jaw tone Y Lateral palpebral N Medial palpebral Y y y Coughing Y Swallowing N Paddling N Vocalization n 230

POST- Lucky IPPV INTO 5 10 15 20 25 30 35 40 45mi 50 Out of Pre-Reco TIVA extub 5min 5 min 10 min 15 min Nov/19/13 Starts MRI min min min min min min min min n min MRI very stop ation post Intub TIME 0830 0835 0840 0845 0850 0855 0900 0905 0910 0945 0953 1000 1006 1012 1017 Fluid Rate P or A rate 70 70-77 84 91 98-115 98 P/A Top Up 1 2 10 HR bpm 81 89 81 86 113 109 98 96 93 92 91 89 90 90 67 68 66 68 73 75 72 7 SAP mmHg 115 115 119 97 96 89 66 62 62 64 63 62 63 62 69 86 82 94 79 75 91 94 DAP mmHg 67 67 70 59 58 53 43 42 42 47 46 42 48 47 53 60 55 54 49 48 56 60 MAP mmHg 80 80 83 69 69 64 52 51 51 52 52 51 52 53 58 67 63 66 58 55 66 70 o Temp C 38.6 37.8 35.4

SPO2 99 99 99 99 99 99 97 98 98 97 98 97 97 97 97 98 99 98

ET CO2 45 47 47 51 55 51 38 38 37 37 36 33 35 36 36 38 40 37 RR 0 1 1 1 12 10 9 9 9 9 8 8 8 8 8 8 10 10 2.8/CI:2 2.1/CI:2.3 LiDCO .9 9 PulseCO 1.4 2.1 2.6 2.9 2.9 1.3 1.2 1.6 2.9 2.1 Anesth score 0 2 3 1 1 0 1 0 0 0 0 0 0 0 0 0 0 0

231

Lucky Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Nov/19/13 Time 0 Time 1006 1012 1017 1022 1027 1042 1057 1112 Score R0 R0 R0 P3 P3 2 1 Other 1103 1114 stand sternal PlasmaSample

Lucky after Before IPPV Out of MRI Nov/19/13 premed Na+ 140 246 145 Cl - 4.2 3.6 3.9 K+ 121 118 114 Hb 15.7 15.2 12.9 pH 7.362 7.134 7.252 PCO2 34.8 51.8 46.5 PaO2 86.3 536 560 HCO3- 15.7 16.6 20.3 ABE -4.6 -12.9 -7 Lactate 1.8 2.8 2 PCV (%) 46 45 37 TP (mg/dL) 68 62 60 BUN 5-15 232

Appendix 2.8: Raw data of dog 8

HR RR TEMP Attitude Date:______Oct 17 2013______Before Instrumentation 120 30 calm Randomization Dog number: _____5______Time Response Attempts Dog ID#: ______EMLA application 0723 Dog Name: ______Major______Cephalic vein 0738 no 1 Meloxicam 0749 (0.46ml) Dog Weight:______23______KG Dorsal pedal artery 0748 no 2 After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______0755______136 108 88 77 4.6 Blood Gas Taken t:_0750_ 146 36 131 96 75 7.49 7.4 146 17.6 Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______0816____ 3.2 Yes No Yes 3 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 0819 Blood Gas Taken :______144 panting 163 110 88 NA 8.6 NA NA Time Starting Induction: ______0821______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______0822______Mid/Sal Volume:______1 2 3 4 5 6 Total dose Intubation score: Injectable Volume Admin Y Y 2.9ml ______2______Attempt to Intubate Y Comments: Relaxed jaw tone N Lateral palpebral Y Medial palpebral Y Coughing Y Swallowing N Paddling N Vocalization N

233

POST- Major INTO 5 Out of Pre-Recove 5 min 10 min 15 min IPPV Starts 10 min 15 min 20 min 25 min 30 min 35 min 40 min 45 min Oct/17/13 MRI min MRI ry Intub 084 TIME 0822 0827 0832 0837 0847 2 Fluid Rate 115 115 115 115 115 115 115 115 115

200 200 200 P or A rate 250 250 250 250 250 250 225 200 200 200 200 200 200 200 200 dopa dopa dopa P/A Top Up

HR bpm 99 83 92 102 111 137 85 95 98 98 98 95 113 117 115 107 113 65 57 57 SAP mmHg 132 113 97 95 97 84 99 72 66 68 71 68 78 85 84 98 100 103 101 101 DAP mmHg 71 69 51 49 50 56 56 39 38 40 40 38 44 47 46 56 56 64 50 52 MAP mmHg 90 79 68 66 68 71 69 49 47 50 50 48 56 60 59 68 70 76 65 66 o Temp C 37.7 36.4

SPO2 98 99 98 98 99 99 99 99 99 99 99 99 99 99 99 99 100 100

ET CO2 28 49 55 66 41 39 39 38 37 37 38 38 39 38 38 36 36 RR 0 0 0 0 13 13 12 12 12 12 12 12 12 12 12 12 13 13 3.94/ LiDCO 1.66/CI:2.06 CI:4.89 6.1;4.2; PulseCO 4.4 5.1 5.8 6.6 6.4 4.2 2.4 2.7 3.3 4.3;4.4

Anesth score 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

234

Major Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/17/13 Time 0 Time 1001 1005 1010 1015 1020 1035 1050 1105 Score R0 R1 R0 P1 P0 P0 P0 Other 1012 1025stand 1055 sternal return to normal PlasmaSample

Major Before Before IPPV Out of MRI Oct/17/13 premed Na+ 146 147 146 Cl - 118 117 116 K+ 3.7 3.2 3.7 Hb 17.6 16.6 13.2 pH 7.393 7.194 7.343 PCO2 31.1 58.9 37.6 PaO2 76.6 514 567 HCO3- 19.6 21.6

ABE -3.2 -7.3 -4.3 Lactate 0.8 20 0.5 PCV (%) 49 46 38 TP (mg/dL) 6.0 5.6 5.3 Bun 5-15 235

HR RR TEMP Attitude Date:______Oct 30_2013______Before Instrumentation 80 48 38.7 BAR Randomization Dog number: ______tx2______Time Response Attempts Dog ID#: ______EMLA application 1230 Dog Name: ______Major______Cephalic vein no 1 Meloxicam 1250,0.5ml Dog Weight:______23______KG Dorsal pedal artery no 2 After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______Blood Gas Taken t:______80 48 164 112 93 6.1 Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: 1350______3.2 No No Yes 2 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 138 159 99 80 9.6 Blood Gas Taken :______133 panting 154 101 73 4.4/CI:5.46 4.2 Time Starting Induction: ______1359______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______1400______Mid/Sal Volume:______1 2 3 4 5 6 Total dose Intubation score: Injectable Volume Admin y y 1.5 ______0______Attempt to Intubate Y(in) _ Relaxed jaw tone Y Comments: Lateral palpebral N Medial palpebral y Coughing N Swallowing N Paddling N Vocalization N

236

POST- IPPV Out Major 5 10 INTO 10 15 20 25 30 35 40 45mi Pre-Recov TIVA extub 5min 15 min Start 5 min of Oct/30/13 min min MRI min min min min min min min n ery stop ation post Intub s MRI TIME 1400 1415 1418 1428 1508 1511 1520 1521 Fluid Rate 250/7 275/ 300/ 350/9 375/1 P or A rate 325/91 0 77 84 8 05 P/A Top Up 1 1 HR bpm 119 105 116 125 131 152 94 91 98 85 97 98 94 92 91 92 88 86 8 81 122 136 SAP mmHg 113 115 94 91 95 92 116 109 105 111 107 108 107 106 107 110 96 100 9 99 102 109 DAP mmHg 56 61 48 49 49 56 76 72 67 74 67 67 67 66 67 69 61 57 54 56 68 63 MAP mmHg 77 79 64 64 64 70 86 81 77 83 79 79 78 77 77 80 69 68 66 67 82 78 o Temp C

SPO2 97 98 98 98 98 97 98 98 99 98 98 98 98 98 98 97 97 97

ET CO2 0 0 0 50 51 38 39 40 39 39 39 39 39 39 38 36 39 RR 0 0 0 7 11 11 11 11 11 11 11 11 11 11 11 11 11 3.98/CI:4. 2.09/CI:2.5 LiDCO 93 9 PulseCO 3.5 2.6 2.7 3.1 3.9 4.1 2.1 2.5 2.3 2.3 3.3 3.1 PulseCO 1:2.2 2:2.5 3:2.7 IND Anesth 1 1 1 1 1 1 0 0 2 1 0 0 0 0 0 1 1 score

237

Major Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/30/13 Time 0 Time 1521 1532 1602 1617 1632 Score R0 R2 R2 P3 O2 P0 P0 Other 1602 1617 stand sternal PlasmaSample

Major after Before IPPV Out of MRI Oct/30/13 premed Na+ 144 146 145 Cl - 116 117 116 K+ 3.6 3.2 3.6 Hb 16.1 16.1 13.4 pH 7.368 7.190 7.355 PCO2 38.6 62.1 40 PaO2 68.9 512 583 HCO3- 21.2 22.4 21.9 ABE -2.5 -6.6 -3.0 Lactate 0.5 0.7 0.6 PCV (%) 46 46 38 TP (mg/dL) 5.6 5.2 5.0 BUN 5-15 238

HR RR TEMP Attitude Date:______Nov 12__2013______Before Instrumentation 132 24 calm Randomization Dog number: ______tx3______Time Response Attempts Dog ID#: ______EMLA application 0730 Dog Name: ______Major______Cephalic vein 0740 no 1 Meloxicam 0814 Dog Weight:______23______KG Dorsal pedal artery 0819 no 4 After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______Blood Gas Taken t:______98 18 130 101 86 3.1 Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: __0825______2.2 No No Yes(after taking temp) 3 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 110 212 115 85 5.2 Blood Gas Taken :______103 24 202 109 81 3.2/CI:3.96 2.9 Time Starting Induction: ______0835______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______0835______Mid/Sal Volume:______1 2 3 4 5 6 Total dose Intubation score: Injectable Volume Admin y y 1.5 ______1______Attempt to Intubate Y(in) Comments: Relaxed jaw tone Y Lateral palpebral N Medial palpebral y Coughing y Swallowing N Paddling N Vocalization N

239

POST- Out Major 5 10 15 IPPV INTO 10 15 20 25 30 35 40 45 Pre-Recov TIVA extub 5min 5 min of Nov/12/13 min min min Starts MRI min min min min min min min min ery stop ation post Intub MRI TIME 0836 0841 0846 0851 0855 0904 0909 0944 0947 0952 0959 1001 1009 1014 Fluid Rate 250/ 300/ 350/ 70-275 84-3 98-3 350/9 P or A rate / 25/9 75/1 8 77 1 05 P/A Top 1 1 1 2 Up HR bpm 118 103 125 140 138 141 150 146 128 124 115 114 115 117 116 117 65 56 60 153 111 SAP mmHg 165 131 108 98 99 99 70 73 79 83 78 77 80 87 87 86 136 116 104 101 83 DAP mmHg 68 58 50 49 49 50 46 47 47 46 43 42 44 49 47 48 60 56 51 50 40 MAP mmHg 89 77 70 66 67 67 54 58 58 60 56 53 56 61 61 61 75 71 65 63 54 o Temp C 37.5 36.9 35.7

SPO2 99 97 99 99 99 99 99 99 99 99 99 99 99 99 99 99 99

ET CO2 42 45 59 61 49 37 36 36 37 37 37 37 38 38 38 36 42 RR 0 0 8 8 12 7 7 7 7 7 7 7 7 7 7 12 11 LiDCO 5.78/7.17 2.09/2.6 PulseCO 4.9 4.2 4.9 4.8 5.1 5.7 2.2 2.1 2.2 3.7 4 PulseCO 3.9 4.3 4.8 IND Anesth 2 1 1 1 3 0 0 0 0 0 0 0 0 0 1 1 score

240

Major Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Nov/12/13 Time 0 Time 1001 1009 1014 1019 1024 1039 1054 1109 Score R0 R0 R2 P3 P3 P1 P0 Other 1044 1104 stand sternal PlasmaSample

Major after Before IPPV Out of MRI Nov/12/13 premed Na+ 146 147 147 Cl - 118 118 117 K+ 3.6 3.2 3.6 Hb 20.4 17.3 13.4 pH 7.345 7.186 7.333 PCO2 39.1 58.6 40 PaO2 71.8 537 592 HCO3- 20.4 21.4 21 ABE -3.8 -8.1 -4.5 Lactate 0.9 1.6 0.8 PCV (%) 46 49 38 TP (mg/dL) 56 56 50 BUN 5-15 241

HR RR TEMP Attitude Date:______Oct 7_2014______Pre-Instrumentation Randomization Dog number: ______Time Response Attempts Dog ID#: ______EMLA application Dog Name: ______Major______Cephalic vein Meloxicam 1102 Dog Weight:______23.7______KG Dorsal pedal artery After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______Blood Gas Taken 2.2/2/2.1/2. t:____ 120 panting 128 105 91 3/2.4/2.2 Fentanyl Admin Volume (mls) Salivation Nausea Defecation Sedation Score Time: 3.2 No No No 3 _____1109______3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 82 30 162 107 87 2.4 BloodGas Taken 4.1/4.2/4.2/ ______94 152 98 75 4.01/CI 4.97 3.9 Time Starting Induction: ______1121______P/A Vol: ______Time: Time: Time: Time: Time: Time: Time Intubation: ______1122______1121 1122 Mid/Sal Vol :______1 2 3 4 5 6 Total dose Intubation score: Injectable Volume X ______0______Attempt to Intubate Comments Relaxed jaw tone Lateral palpebral Medial palpebral Coughing x Swallowing Paddling Vocalization

242

5 min POST- IPPV Out Major INTO Pre-Recove TIVA post 5 min 10min 15 min Start 5 min 10 min 15 min 20 min 25 min 30 min 35 min 40 min of Oct/07/14 MRI ry OFF extuba Intub s MRI tion TIME 1122 1127 1132 1137 1145 1153 1158 1203 1208 1213 1218 1223 1228 1233 1236 1243 1247 1256 Fluid Rate

250/70- 300/8 325/9 P or A rate 350/98 275/77 4 1 1123 P/A Top Up# 1133 1145 1151 1125 HR bpm 84 71 89 97 93 112 104 111 111 113 117 114 116 114 122 80 61 65 59 68 SAP mmHg 129 120 117 101 98 106 92 87 90 90 91 95 102 103 100 120 111 109 108 126 DAP mmHg 66 56 59 54 52 60 59 54 57 54 54 57 62 64 62 64 56 56 55 63 MAP mmHg 85 76 73 67 65 74 71 66 70 67 68 70 77 79 76 79 71 71 71 78 o Temp C

SPO2 95 99 98 99 98 99 99 99 99 99 99 99 99 95 95 96

ET CO2 46 44 50 41 40 41 41 42 43 43 44 45 45 40 38

RR 0 13 13 11 10 10 10 10 10 10 10 10 10 10 10 10

3.24/CI LiDCO 4.02 4.3 3.1/3. 2/1. 4.5/4. PulseCO 4 3.1/3.2 /4. 3/3.3 3.1/3 9/1. 4 4 /3.4 8 3.1/3. 3.2/3.3/ 3.4/3.3 3.2 PulsCO IND 2 3.4 1min 2min 3min 4 min Anesth score 1 0 1 0 1 1 0 0 0 0 0 0 0 0 0 0

243

Major Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/07/14 Time 0 Time 1247 1251 1256 1301 1306 1321 1336 1351 Score R0 R0 R0 P3 P2 P0 P0 Other Sternal 1215 Stand 1314

PlasmaSample

Major 3min after Pre-Vent End MRI Oct/07/14 fentanyl Na+ 147 148 148 Cl - 119 118 117 K+ 3.2 3 3.4 Hb 16.8 15.1 13.3 pH 7.402 7.244 7.311 PCO2 32.5 51.7 44.4 PaO2 105 481 577 HCO3- 19.5 21.5 22.1 ABE -3.2 -5.9 -4 Lactate 0.7 0.7 0.7 PCV (%) 48 44 38 TP (mg/dL) 60 60 58 Plasma Sample:

244

HR RR TEMP Attitude Date:______Oct 15_2014______ Pre-Instrumentation 96 20 38.2 Good Randomization Dog number: ______Time Response Attempts Dog ID#: ______EMLA application 900 Dog Name: ______Major______Cephalic vein Meloxicam 950 Dog Weight:______23.5______KG Dorsal pedal artery Rt. Poor, 24G After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time:______Blood Gas Taken t:______126 36 123 99 88 2.5/2.6 Fentanyl Admin Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______1027______3.3 Yes/No Yes/No Yes/No 3 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 89 16 170 111 84 3.1/2.8 BloodGas Taken 91 17 173 118 93 2.6/2.5 ______91 172 111 84 3.29 CI 4.08 3.1/3.2/3.4 Time Starting Induction: _____1037______P/A Time: Time: Time: Time: Time: Time: Time Intubation: ______1037______Vol: ______1037 1037 Mid/Sal 1 2 3 4 5 6 Total dose Intubation score: Vol :______2______Injectable Volume Comments Attempt to Intubate x Relaxed jaw tone Lateral palpebral Medial palpebral Coughing Swallowing Paddling Vocalization

245

5 min POST- Out TIV Major 10 IPPV INTO Pre-Recove post 5 min 15 min 5 min 10 min 15 min 20 min 25 min 30 min 35 min 40 min of A Oct/15/14 min Starts MRI ry extuba Intub MRI OFF tion TIME 1037 1042 1047 1052 1158 1108 1113 1118 1123 1128 1133 1138 1143 1148 1155 1203 1205 1216 Fluid Rate

250/70- P or A rate 275/77 P/A Top Up# 1039

HR bpm 93 56 71 86 89 101 99 113 107 100 92 95 89 102 104 61 55 58 57 70 SAP mmHg 128 112 106 109 103 97 105 82 89 86 82 82 84 83 89 118 105 103 111 98 DAP mmHg 66 58 58 59 56 51 48 54 61 60 57 56 59 57 60 52 47 48 50 51 MAP mmHg 89 74 72 72 72 69 67 65 70 69 65 65 67 67 71 68 61 63 65 64 o Temp C

SPO2 96 96 95 96 97 98 98 99 100 100 100 100 100 100 97 96 96 95

ET CO2 30 20 19 20 50 37 38 38 393 9 39 40 40 40 40 38 38 RR 0 0 0 0 11 12 12 12 12 12 12 12 12 12 12 12 12 LiDCO 4.57 CI 5.6 1.83 CI 2.27

4.5/ 5/5.2/ 2.3/2. 2.2/ 1.8/ 1.7/1. 2.5/2. 3.5/3. 4.6/ 1.7/1. 2/2.1/2. PulseCO 2.9/3 5.6/5. 4/2.5/ 2.3/ 1.9/ 8/1.9 7/2.8 6/3.8 4.7/ 9/2 2/2.3 7 2.6 2.4 2 4.8

1.7/1. 1.6/1.7/ 1.7/1.8 1.7/1. PulsCO IND 8/1.9 1.9 1min 8 3min 4 min 2min Anesth score 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

246

Major Stop TIVA Extub E 5 E 10 E 15 /14E 45 E 60 E90 E 120 Oct/15/14 Time 0 Time 1205 1211 1216 1221 1226 1256 1311 Score R0 R0 R0 P3 P0 P0 Other Stand 1254

PlasmaSample

Major 3min after Pre-Vent End MRI Oct/15/14 fentanyl Na+ 147 148 146 Cl - 118 120 116 K+ 3.2 2.8 3.6 Hb 17 15 13 pH 7.379 7.183 7.274 PCO2 33.2 53.6 46 PaO2 76.7 399 533 HCO3- 19.2 19.4 21 ABE -4.5 -9.3 -5.8 Lactate 0.6 0.8 0.4 PCV (%) 48 43 38 TP (mg/dL) 60 54 54 Plasma Sample:

247

Appendix 2.9: Raw data of dog 9

HR RR TEMP Attitude Date:______Oct 23 2013 p.m.______Before Instrumentation 90 18 38.5 calm Randomization Dog number: ______10______Time Response Attempts Dog ID#: ______EMLA application 1000 Dog Name: ______Mystique______Cephalic vein 1 no 5, Lt cephalic Meloxicam 1114, 0.51ml Dog Weight:______25______KG Dorsal pedal artery 1135 After 30 min rest1205 HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Blood Gas :______69 18 127 100 86 NA 3.2/2.9 Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______1214_____ 3.6ml No No No 3 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 46 13 143 93 92 1.7/1.6 Blood Gas Taken :______56 163 100 80 2.4/2.6 72 42 155 109 88 3.05/CI:3.6 3.1/3/3 148 15.4 Time Starting Induction: ______1225______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______1227___ Mid/Sal Volume:______1 2 3 4 5 6 Total dose Intubation score: 2 Injectable Volume y y y 2.1ml ______Attempt to Intubate Y y Comments: Relaxed jaw tone y Y Lateral palpebral y N Medial palpebral y Coughing N n Swallowing y Y Paddling N N Vocalization n n

248

POST- Mystique 10 IPPV INTO Out of Pre-Recove 5 min 15 min 5 min 10 min 15 min 20 min 25 min 30 min 35 min 40 min 45 min Oct/23/13 min Starts MRI MRI ry Intub TIME 1227 1232 1237 1242 1246 1255 1300 1305 1310 1315 1300 1325 1330 1335 1340 1343 1349 Fluid Rate 130 130 130 130 130 130 130 130 130 130

98 98 98 98 98 P or A rate 70 77 84 84 91 91 91 98 98 98 98 98 D:5 D:5 D:10 D:10 D:10 P/A Top Up 2 2 1346

HR bpm 109 109 144 145 147 141 153 121 113 107 106 106 101 90 78 60 65 69 71 SAP mmHg 112 116 105 100 100 93 81 75 74 75 75 75 76 84 92 97 98 99 97 DAP mmHg 65 64 61 59 64 55 51 48 46 47 47 47 45 49 49 47 52 58 58 MAP mmHg 80 77 75 71 71 71 60 56 54 56 55 55 54 59 60 60 60 70 66 o Temp C

SPO2 95 97 97 97 98 98 98 98 99 99 99 99 99 99 98 99 97

ET CO2 38 43 50 58 53 42 41 40 39 39 39 39 39 39 39 45 43 RR 0 0 0 9 10 10 10 10 10 10 10 10 10 10 10 10 10 LiDCO 3.82/4.51

PulseCO 4.2 4.3 5.5 3.1 3.2 3.9/4 Anesth score 1 3 1 1 3 2 1 0 0 0 0 0 0 0 0 2 1

249

Mystique Stop Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/23/13 TIVA Time 0 Time 1353 1359 1404 1409 1414 1429 1444 1459 Score R0 R2 R2 R2 P3 P1 P0 Other 1441 1454 sternal standing/ambulate PlasmaSample

Mystique Post-fentanyl Before IPPV Out of MRI Oct/23/13 Na+ 148 149 148 Cl - 117 3.2 3.7 K+ 3.5 117 116 Hb 15.4 16.7 13.1 pH 7.311 7.165 7.278 PCO2 40.5 62.1 47.6 PaO2 68 452 524 HCO3- 19.4 21.2

ABE -5.5 -8.4 -4.2 Lactate 0.5 1.1 0.4 PCV (%) 45 46 38 TP (mg/dL) 6.2 6.2 5.8 BUN 5-15 250

HR RR TEMP Attitude Date:______Nov5______Before Instrumentation 92 16 38.4 Randomization Dog number: ______tx2______Time Response Attempts Dog ID#: ______EMLA application 0910 Dog Name: ______Mystique______Cephalic vein 0920 n 1 Meloxicam 1030 Dog Weight:______25______KG Dorsal pedal artery 0930 1 After 30 min :1030 HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Blood Gas Taken t:__ 72 20 120 95 81 1.5 Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______1043___ 3.5 No No No 2 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 62 18 124 93 78 1.4 Blood Gas Taken :__ 99 18 158 108 85 2.8 84 18 165 113 90 2.88/3.4 2.5 Time Starting Induction: ___1055______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______Mid/Sal Volume:______1 2 3 4 5 6 Total dose Intubation score:2 Injectable Volume Admin y y y y y y ______Attempt to Intubate y Comments: Relaxed jaw tone n n n y y Lateral palpebral y y y y n Medial palpebral y y y y Y Coughing Y Swallowing Y Paddling Vocalization

251

POST- Mystique IPPV INTO Out of Pre-Recov TIVA extuba 5min 5 min 10 min 15 min 5 min 10 min 15 min 20 min 25 min 30 min 35 min Nov/05/13 Starts MRI MRI ery stop tion post Intub TIME 1057 1112 1118 1141 1146 1216 1219 1225 1230 1236 1241 Fluid Rate 350/98- 375/ 425/ 475/13 250/70- 300/84- 500/14 525/14 P or A rate 105-400 119-450/ 3-500/ 275/77 325/91 0 7 / 126 140 112 P/A Top Up 2 2 5 1 1 1 13 13 HR bpm 85 87 130 139 88 77 77 74 95 79 56 61 70 69 68 58 82 69 6 3 10 10 SAP mmHg 122 105 99 99 94 84 86 85 120 137 113 112 106 96 96 100 109 113 1 5 DAP mmHg 66 55 54 59 63 59 58 52 53 54 84 87 66 65 61 53 54 50 61 60 MAP mmHg 84 70 70 72 71 71 67 62 62 63 96 101 80 78 73 65 66 64 72 74 o Temp C 37.2 35.9

SPO2 98 97 99 97 98 98 97 97 97 97 97 97 97 97 97 98

ET CO2 0-29 27 39 40 44 36 40 40 40 40 42 37 38 37 35 34 RR 0-14 11 19 14 11 11 11 11 11 11 10 10 10 10 10 10 5.55/6.5 LiDCO 2.35/2.78 5 PulseCO 3.1 3.8 5.2 6.1 6.6 6.8 2.6 2.5 2.6 2.3 .27 2.6 PulseCO IND 3.5 3.3 3.6 Anesth score 3 3 4 2 3 1 0 0 0 1 0 0 1 1

252

Mystique Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Nov/05/13 Time 0 Time 1230 1236 1241 1246 1251 1306 1321 1336 Score R0 R2 R2 P2 P0 P0 Other Paddling 1300 sternal 1306 stand PlasmaSample

Mystique after Before IPPV Out of MRI Nov/05/13 premed Na+ 150 150 146 Cl - 119 118 117 K+ 3.6 3.4 3.9 Hb 14.3 16.9 7.309 pH 7.317 7.237 41.7 PCO2 38.1 47.5 608 PaO2 73.2 577 HCO3- 19.5 ABE -6.2 -6.7 -4.7 Lactate 0.6 1.5 0.7 PCV (%) 42 49 37 TP (mg/dL) 60 62 58 BUN 5-15 253

HR RR TEMP Attitude Date:______Nov 18______Before Instrumentation 104 pant 38.2 BAR Randomization Dog number: ______tx2______Time Response Attempts Dog ID#: ______EMLA application 0900 Dog Name: ______Mystique______Cephalic vein n 1 Meloxicam 0957;0.5ml Dog Weight:______25______KG Dorsal pedal artery n 1 After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Blood Gas Taken t:__ 104/72 24 130/135 98/91 80/73 3.4/2.6 Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______0959___ 3.5 y No No 1 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 95 141 112 89 2.3 Blood Gas Taken :__ 88 159 108 84 3.9 93 15 165 107 87 3.5/CI:3.98 3.8 1043 P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______Mid/Sal Volume:______1 2 3 4 5 6 Total dose Intubation score: Injectable Volume Admin y y y 2.3ml ______1______Attempt to Intubate Y Comments: Relaxed jaw tone Y Lateral palpebral Y y Y Medial palpebral y Y y Coughing Swallowing Paddling Vocalization

254

POST- Out Mystique IPPV INTO 5 10 15 20 25 30 35 40 45mi 50 55 Pre-Recov extub 5min 5 min 10 min 15 min of Nov/19/13 Starts MRI min min min min min min min min n min min ery ation post Intub MRI TIME 1044 1049 1054 1059 1104 1110 1115 1205 1211 1216 1223 1228 Fluid Rate 250/70- 350/98- 425/11 450/ 450/ 475/ P or A rate ---325/9 --400/1 425/119 9 126 126 133 1 12 P/A Top Up 3 2 1 3 1 15 13 HR bpm 125 139 138 138 150 131 130 130 122 112 107 106 109 102 85 75 72 60 60 71 70 2 9 10 SAP mmHg 120 105 104 99 95 85 88 81 88 82 79 87 81 85 93 101 108 123 100 110 101 103 1 DAP mmHg 70 53 56 55 56 54 49 55 52 53 51 52 51 54 55 58 63 66 72 70 56 48 57 MAP mmHg 87 71 72 71 71 68 63 67 63 67 62 62 61 62 65 69 72 76 83 54 76 62 74 o Temp C 35.8

SPO2 96 96 96 98 98 99 99 99 99 99 99 99 99 98 98 98 99 98 98

ET CO2 39 41 48 47 49 40 37 37 37 37 37 36 36 36 36 36 36 39 41 RR 0 9 6 8 10 8 8 8 8 8 8 8 8 8 8 8 8 8 8 LiDCO 2.2/2,51 6. PulseCO 4.7 6.3 5.4 3.4 3.2 1.07 1.1 2.1 3.8 3.1 2 PulseCO IND 4.5 5.6 6.2 Anesth score 3 3 1 1 1 3 0 0 0 0 0 0 0 0 1 0 0 1 0

255

Mystique Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Nov/19/13 Time 0 Time 1220 1223 1228 1233 1238 1253 1308 1323 Score R0 R0 R0 P3 P3 P2 P1 Other twitching twitching twitching calm PlasmaSample

Mystique after Before IPPV Out of MRI Nov/19/13 premed Na+ 147 148 147 Cl - 3.6 3.2 3.7 K+ 118 119 117 Hb 15.3 16.7 12.9 pH 7.303 7.259 7.317 PCO2 40.1 38 40.7 PaO2 79.9 603 554 HCO3- 19.4 ABE -6.1 -9.3 -4.7 Lactate 1.2 3.2 1.5 PCV (%) 44 48 37 TP (mg/dL) 60 58 56 BUN 5-15 256

Appendix 2.10: Raw data of dog10

HR RR TEMP Attitude Date:______Oct 24_2013______Before Instrumentation 90 36 38.6 calm Randomization Dog number: ______11______Time Response Attempts Dog ID#: ______EMLA application 0725 Dog Name: ______Ruby______Cephalic vein 0740 no 1 Meloxicam 0742 Dog Weight:______17.5______KG Dorsal pedal artery 0745 no 2 After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Blood Gas Taken t:______102 24 102 91 78 NA 5.5/5.8 NA NA Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______0810_____ 2.5 Yes Yes Yes 1 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 73 54 124 75 64 Blood Gas Taken :______133 Panting 156 103 80 121 panting 175 98 79 5.04/7.63 2.8/4.8 Time Starting Induction: ______0827______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______0829______Mid/Sal Volume:______1 2 3 4 5 6 7 Total dose Intubation score: Injectable Volume Admin y y y y y y y ______3______Attempt to Intubate y Y(in) Comments: Relaxed jaw tone Lateral palpebral y y Y n n Medial palpebral y y y Y y Coughing y Y Swallowing Paddling Vocalization

257

POST- Out Ruby IPPV INTO Pre-Reco extub 5min 5 min 10 min 15 min 5 min 10 min 15 min 20 min 25 min 30 min 35 min 40 min of Oct/24/13 Starts MRI very ation post Intub MRI TIME 0830 0852 0858 0938 0944 0952

Fluid Rate

70/250- 84/300- P or A rate 98/350 77/275 91/325 P/A Top Up 2 1 1

HR bpm 85 88 87 86 80 85 84 85 84 77 74 73 69 72 71 60 54 58 60 56 SAP mmHg 96 94 92 85 85 82 81 74 74 79 80 81 81 80 78 97 95 91 89 93 DAP mmHg 55 56 51 49 47 47 44 48 48 47 48 47 45 45 45 57 51 50 46 52 MAP mmHg 67 66 62 58 56 57 54 55 55 56 56 57 55 55 54 65 63 61 60 61 o Temp C

SPO2 97 97 98 99 99 98 99 99 99 99 99 99 99 99 95 95 95 95

ET CO2 34 46 46 55 47 36 36 37 36 35 35 35 35 35 32 34 RR 19 10 12 8 10 10 10 10 10 10 10 10 10 10 10 10 2.12/3. LiDCO 22 3. PulseCO 4.1 3.6 3.8 3.1/2.4 2.2 2.0 1.6 4 1:4.0/3. PulseCO IND 2:3.5 3:3.5 5/3.8 Anesth score 2 1 2 1 1 2 1 1 1 1 1 1 1 1 1 1

258

Ruby Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Oct/24/13 Time 0 Time 0955 1001 1031 1046 1101 Score R0 R0 R0 P3 P1 P0 P0 Other 102 1046 6sternal standing/ ambulate PlasmaSample

Ruby after Before IPPV Out of MRI Oct/24/13 premed Na+ 146 147 146 Cl - 118 119 117 K+ 3.6 3.3 3.8 Hb 16.4 16.5 14.0 pH 7.366 7.230 7.351 PCO2 35.9 49.9 37 PaO2 85.2 538 546 HCO3- 19.7 20.2

ABE -3.8 -7.6 -4.6 Lactate 1.0 1.9 0.8 PCV (%) 46 47

TP (mg/dL) 5.6 5.4

BUN 5-15 259

HR RR TEMP Attitude Date:______Nov6_2013______Before Instrumentation 126 54 39.2 Randomization Dog number: ______tx2______Time Response Attempts Dog ID#: ______EMLA application 0845 Dog Name: ______Ruby______Cephalic vein 0845 n 1 Meloxicam 0920 Dog Weight:______18______KG Dorsal pedal artery 0934 3 After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Blood Gas Taken t:__ 122 24 118 73 59 2 Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______0937___ 2.5 n n n 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 79 48 166 98 75 5.4 Blood Gas Taken :__ 89 115 87 71 2.36/3.44 2.2 84 122 71 51 1.99/2.89 2.5 Time Starting Induction: ___0951______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______Mid/Sal Volume:______1 2 3 4 5 6 Total dose Intubation score:2 Injectable Volume Admin y Y ______1______Attempt to Intubate Y y Comments: Relaxed jaw tone Y Lateral palpebral Y N Medial palpebral y Y Coughing y Swallowing Paddling Vocalization

260

POST- Ruby IPPV INTO 10 15 20 25 30 35 40 45 Out of Pre-Reco TIVA extub 5min 5 min 10 min 15 min 5 min Nov/05/13 Starts MRI min min min min min min min min MRI very stop ation post Intub TIME 0952 0957 1002 1007 1011 1020 1025 1105 1111 1116 1121 1131 1136 Fluid Rate 325/9 250/70- 375/1 400/1 P or A rate 300/84 1-350/ 275/77 05 12 98 P/A Top Up 1 1 1 HR bpm 120 84 87 87 92 89 74 88 83 86 90 89 94 106 110 107 62 61 58 75 70 SAP mmHg 105 102 93 92 90 85 68 86 87 88 92 105 124 140 140 142 112 111 105 104 100 DAP mmHg 55 55 51 51 50 52 52 59 59 62 65 72 85 92 94 95 67 65 62 61 59 MAP mmHg 70 68 62 63 60 60 58 68 68 69 73 82 96 104 107 110 81 75 75 74 71 o Temp C 37.5

SPO2 95 98 98 99 98 98 98 98 98 97 97 97 97 97 97 98 98

ET CO2 38 50 53 54 40 40 41 40 40 42 43 44 40 39 39 33 34 RR 20 9 6 6 10 10 10 10 9 9 10 10 10 10 10 10 10 2.18/3. LiDCO 1.36/1.97 17 2. 0.9 PulseCO 4.1 2.0 2.3 2.4 1..6 1 1.07 1.91 1.6 4 6 PulseCO IND 3.1 2.4 2.3 Anesth score 2 1 1 1 1 1 0 0 0 0 1 1 0 0 0 1

261

Ruby Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Nov/05/13 Time 0 Time 1121 1131 1136 1141 1146 1201 1216 1231 Score R0 R0 R0 P3 P3 P3 Other 1231sternal 1247 stand PlasmaSample

Ruby after Before IPPV Out of MRI Nov/05/13 premed Na+ 146 147 146 Cl - 117 118 117 K+ 3.6 3.2 3.6 Hb 15.3 14.6 14.9 pH 7.352 7.201 7.358 PCO2 37.9 56.8 34.6 PaO2 67.5 497 600 HCO3- ABE -3.9 -7.1 -5.4 Lactate 0.7 0.7 0.9 PCV (%) 43 40 42 TP (mg/dL) 54 50 48 BUN 5-15 262

HR RR TEMP Attitude Date:______Nov19_2013______Before Instrumentation 130 20 38.8 QAR Randomization Dog number: ______tx3______Time Response Attempts Dog ID#: ______EMLA application 1130 Dog Name: ______Ruby______Cephalic vein n 1 Meloxicam 1230; 0.35ml Dog Weight:______18______KG Dorsal pedal artery n 2 After 30 min rest HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Blood Gas Taken t:__ 112 20 119 97 84 2.9 Fentanyl Administration Volume (mls) Salivation Nausea Defecation Sedation Score Time: ______2.5 n n n 3 3 min after premed HR RR SAP MAP DAP LiDCO PulseCO Na+ Hgb Time: 58 24 123 99 78 3.27 2.6 Blood Gas Taken :__ 81 121 99 76 4.76 2.2 108 125 86 66 4.5 4.2 Time Starting Induction: ______P/A Volume : ______Time: Time: Time: Time: Time: Time: Time Intubation: ______Mid/Sal Volume:______1 2 3 4 5 6 Total dose Intubation score: Injectable Volume Admin y Y 2.8ml ______1_____ Attempt to Intubate y Comments: Relaxed jaw tone Lateral palpebral Medial palpebral Coughing Swallowing Paddling Vocalization

263

POST- Out Ruby IPPV INTO 10 15 20 25 30 35 40 45 50 Pre-Recov TIVA extub 5min 5 min 10 min 15 min 5 min of Nov/19/13 Starts MRI min min min min min min min min min ery stop ation post Intub MRI TIME 0150 0155 0200 0205 0210 0215 0220 0303 0310 0313 0318 0323 0328 Fluid Rate P or A rate 250 275 P/A Top Up 1 HR bpm 67 67 62 71 80 60 66 69 61 60 64 67 65 66 64 55 57 58 77 SAP mmHg 106 96 91 83 96 95 96 100 104 102 102 102 102 101 103 102 100 97 136 DAP mmHg 57 61 48 46 46 58 61 63 62 62 61 61 60 61 59 58 56 52 71 MAP mmHg 70 70 62 56 67 68 70 72 73 71 70 70 71 70 71 69 67 64 88 o Temp C 38 37.6 37.6

SPO2 99 99 99 99 99 99 99 99 97 99 99 99 99 99 99 98 98

ET CO2 38 38 39 52 47 37 37 34 31 34 35 36 36 36 36 43 46 RR 1 2 8 8 8 8 7 7 10 10 10 10 10 10 10 10 LiDCO 1.9/2.77 PulseCO 1.8 1.7 1.7 1.8 2.2 1.2 1.4 1.6 2.3 1.2 PulseCO IND 1.7 2.1

Anesth score 1 1 0 1 1 0 0 0 0 0 0 0 0 0 0 0 1 1

264

Ruby Stop TIVA Extub E 5 E 10 E 15 E 30 E 45 E 60 E90 E 120 Nov/19/13 Time 0 Time 0318 0323 0328 0333 0338 0353 1608 1623 Score R0 R2 R0 P3 P3 Other 1557 sternal PlasmaSample

Ruby after Before IPPV Out of MRI Nov/19/13 premed Na+ 147 147 146 Cl - 3.2 119 3.3 K+ 117 2.9 116 Hb 15.1 15.3 13 pH 7.323 7.266 7.25 PCO2 35.8 40.1 47.1 PaO2 72.8 586 568 HCO3- 19.1 ABE -6.9 -8 -5.7 Lactate 0.5 0.5 0.4 PCV (%) 42 44 38 TP (mg/dL) 54 50 48 BUN 5-15 265

Appendix 3: Treatments assignment Dog Name Date Imaging Modalities Treatment 1 Alonzo 101414 MRI AS 1 Alonzo 100614 CT2 PS 2 Baby 101713 MRI PS 2 Baby 103113 CT1 PM 2 Baby 111213 MRI AM 2 Baby 101514 MRI PM 2 Baby 100714 CT2 AM 3 Baron 101613 MRI PS 3 Baron 103013 CT1 AS 3 Baron 111113 MRI PM 3 Baron 101514 MRI AS 3 Baron 100814 CT2 PM 4 Bolt 101513 MRI PM 4 Bolt 102813 CT1 PS 4 Bolt 110813 MRI AS 4 Bolt 101414 MRI AM 4 Bolt 100614 CT2 PM 5 Chance 101513 MRI AS 5 Chance 102813 CT1 AM 5 Chance 110813 MRI PS 6 Hunter 101613 MRI AM 6 Hunter 103013 CT1 AS 6 Hunter 111113 MRI PS 7 Lucky 102313 MRI AS 7 Lucky 110513 CT1 PM 7 Lucky 111913 MRI AM 8 Major 101713 MRI PM 8 Major 103013 CT1 AM 8 Major 111213 MRI AS

266

8 Major 101514 MRI PS 8 Major 100714 CT2 AS 9 Mystique 102313 MRI AM 9 Mystique 110513 CT1 PS 9 Mystique 121813 MRI AS 10 Ruby 102413 MRI PS 10 Ruby 110613 CT1 AM 10 Ruby 111913 MRI PM

267